Dengue vaccine unit dose and administration thereof

ABSTRACT

The invention relates to a unit dose of a dengue vaccine composition and methods and uses for preventing dengue disease and methods for stimulating an immune response to all four dengue virus serotypes in a subject or subject population. The unit dose of a dengue vaccine composition includes constructs of each dengue serotype, such as TDV-1, TDV-2, TDV-3 and TDV-4, at various concentrations in order to improve protection from dengue infection.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. § 371 United States National PhaseApplication of, and claims priority to PCT Application No.PCT/US2019/049749, filed Sep. 5, 2019, which is a Continuation of U.S.application Ser. No. 16/295,611, filed on Mar. 7, 2019. PCT ApplicationNo. PCT/US2019/049749, claims the benefit of priority from EuropeanApplication No. 19161184.7 filed on Mar. 7, 2019, European ApplicationNo. 19154334.7 filed on Jan. 29, 2019, European Application No.18192701.3 filed on Sep. 5, 2018, European Application No. 18192711.2filed on Sep. 5, 2018, European Application No. 18192717.9 filed on Sep.5, 2018, European Application No. 18192776.5 filed on Sep. 5, 2018,European Application No. 18192787.2 filed on Sep. 5, 2018, EuropeanApplication No. 18192793.0 filed on Sep. 5, 2018, European ApplicationNo. 18192800.3 filed on Sep. 5, 2018, European Application No.18192814.4. The entire contents of these applications are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which is submittedherewith in electronically readable ASCII format via EFS-Web, and ishereby incorporated by reference in its entirety. The electronicSequence Listing file was created on Mar. 2, 2021, is named“2021-03-02_T08269WO1US-SeqListing_ST25.txt” and is 345 KB in size.

FIELD OF THE INVENTION

The present invention relates to a safe and effective method ofinoculation against dengue disease and a corresponding safe andeffective dengue vaccine. In particular the present invention relates toa safe and effective method of inoculation against dengue diseaseirrespective of serostatus and a corresponding safe and effective denguevaccine. The present invention relates also to unit doses of a denguevaccine composition and methods for administering a unit dose of adengue vaccine composition to a subject or a subject population in abroad age group. The present invention is also related to particularconcomitant administration regimes, wherein the unit does/vaccinecomposition is administered concomitantly with one or more of: a yellowfever (YF) vaccine, a hepatitis A vaccine, a human papillomavirus (HPV)vaccine, a combined measles, mumps and rubella (MMR) vaccine, a combinedtetanus, diphtheria, and pertussis (whooping cough) (Tdap) vaccine,and/or a combined vaccine for diphtheria, tetanus, pertussis,poliomyelitis and Haemophilus influenzae type b (DTap/IPV/Hib), or anycombination of the concomitant administration regimes mentioned above.The unit dose according to this invention provides immune responsesagainst all serotypes of dengue virus, i.e. DENV-1, DENV-2, DENV-3 andDENV-4.

BACKGROUND OF THE INVENTION

Vaccines for protection against viral infections have been effectivelyused to reduce the incidence of human disease. One of the mostsuccessful technologies for viral vaccines is to immunize animals orhumans with a weakened or attenuated virus strain (a “live attenuatedvirus”). Due to limited replication after immunization, the attenuatedvirus strain does not cause disease. However, the limited viralreplication is sufficient to express the full repertoire of viralantigens and can generate potent and long-lasting immune responses tothe virus. Thus, upon subsequent exposure to a pathogenic virus strain,the immunized individual is protected from the disease. These liveattenuated viral vaccines are among the most successful vaccines used inpublic health.

Dengue disease is a mosquito-borne disease caused by infection with adengue virus. Dengue virus infections can lead to debilitating andpainful symptoms, including a sudden high fever, headaches, joint andmuscle pain, nausea, vomiting and skin rashes. To date, four serotypesof dengue virus have been identified: dengue-1 (DENV-1), dengue-2(DENV-2), dengue-3 (DENV-3) and dengue-4 (DENV-4). Dengue virusserotypes 1˜4 can also cause dengue hemorrhagic fever (DHF) and dengueshock syndrome (DSS). In the most severe cases, DHF and DSS can be lifethreatening. Dengue viruses cause 50-100 million cases of debilitatingdengue fever, 500,000 cases of DHF/DSS, and more than 20,000 deaths eachyear, a large portion of which are children. All four dengue virusserotypes are endemic throughout the tropical regions of the world andconstitute the most significant mosquito-borne viral threat to humansthere. Dengue viruses are transmitted to humans primarily by Aedesaegypti mosquitoes, but also by Aedes albopictus mosquitoes. Infectionwith one dengue virus serotype results in life-long protection fromre-infection by that serotype, but does not prevent secondary infectionby one of the other three dengue virus serotypes. In fact, previousinfection with one dengue virus serotype may lead to an increased riskof severe disease (DHF/DSS) upon secondary infection with a differentserotype.

To date, only one vaccine, a tetravalent dengue vaccine based on ayellow fever backbone, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon,France), has been licensed in several countries based on the clinicaldemonstration of an overall vaccine efficacy (VE) againstvirologically-confirmed dengue (VCD) of 56-61% in children in Asia andLatin America (Capeding M R et al. Clinical efficacy and safety of anovel tetravalent dengue vaccine in healthy children in Asia: a phase 3,randomised, observer-masked, placebo-controlled trial. Lancet 2014,384:1358-65; Villar L A et al. Safety and immunogenicity of arecombinant tetravalent dengue vaccine in 9-16 year olds: a randomized,controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013,32:1102-9). However, clinical trials have shown that Dengvaxia® canenhance, rather than reduce, the risk of severe disease due to dengueinfection in individuals who had not been previously infected by adengue virus (seronegative populations). Therefore, Dengvaxia® is onlyrecommended for use in individuals who had been previously infected withat least one dengue virus serotype (seropositive populations). Morespecifically, according to the European Medicine Agencys European PublicAssessment report (EPAR) for the product, Dengvaxia® is only for use inpeople from 9 to 45 years of age who have been infected with denguevirus before and who live in areas where this infection is endemic.Endemic areas are areas where the disease occurs regularly throughoutthe year. See also Sridhar S et al. Effect of Dengue Serostatus onDengue Vaccine Safety and Efficacy. N Engl J Med 2018, 379:327-40; andWorld Health Organization. Dengue vaccine: WHO position paper—September2018. Wkly. Epidemiol. Rec. 2018, 93:457-476. S. R. Hadinegoro et al.report in the New England Journal of Medicine, Vol. 373, page 1195, in“Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of EndemicDisease” a pooled risk of hospitalization for virologically-confirmeddengue disease among those under the age of 9 years of 1.58 indicatingan increased risk for the vaccinated group with respect to severedengue. This leaves a substantial unmet need for an effective vaccinewith a good safety profile in both dengue-naïve and seropositiveindividuals, including those dengue-naïve populations living in endemicareas, younger individuals who may not have developed any seropositiveresponse to dengue or been exposed to dengue, and travelers andindividuals from non-endemic regions. There is also a need for outbreakcontrol or travel vaccination, offering a reduction in the risk ofdengue after only one dose.

One further disadvantage of the only currently approved dengue vaccine,Dengvaxia®, is that it must only be given to people who have had apositive test result showing a previous infection with dengue virus(EPAR), i.e. individuals with known serostatus for dengue. Thus,individuals with unknown serostatus for dengue cannot be vaccinated withDengvaxia®.

There is hence a need for a dengue vaccine and corresponding method ofinoculation that stimulates an immune response to all dengue serotypes,preferably a balanced immune response to all serotypes, and protectsagainst dengue disease of any severity (including DSS, DHF), both inseronegative and seropositive populations, which is safe for a largergroup of ages, in particular also for subjects of 9 years and younger.The development of a safe and effective vaccine capable of protectingall populations, including both seronegative and seropositivepopulations, and in particular children and young adults and elderlysubjects in endemic settings and for the purpose of traveling,represents an important approach to the prevention and control of thisglobal disease.

There is thus a medical need for a dengue vaccine and correspondingmethod of inoculation which, as well as being safe and efficaciousirrespective of serostatus and in a broad age group. There is a need fora dengue vaccine and corresponding method of inoculation that avoidscostly and time consuming serostatus tests or seroprevalenceconsiderations. There is a need for a dengue vaccine and correspondingmethod of inoculation that can be used in an outbreak situation.Furthermore there is a medical need for a dengue vaccine which as wellas being safe and effective can also be administered to individuals withunknown dengue serostatus, children under 9 years and seronegativeindividuals.

There is also a need for a vaccine that is administered in fewer dosesthan the current Dengvaxia® dosing schedule of 3 doses, 6 months apart,such as a vaccine that can be administered in only two doses or one doseto be efficacious.

The above objects are commensurate with the research priorities providedby the WHO in the Dengue Vaccine: WHO position paper—September 2018(Wkly. Epidemiol. Rec. 2018, 93:457-476).

Yellow fever (YF) is an acute viral hemorrhagic disease transmitted byinfected mosquitoes of the Aedes aegypti specie. Symptoms of yellowfever take 3 to 6 days to develop and include fever, headache, jaundice,muscle pain, nausea, vomiting and fatigue. A small proportion ofpatients (about 15% of people) who contract the virus develop a severedisease that can lead to bleeding, shock, organ failure, and sometimesdeath. The virus is endemic in tropical areas of Africa and Central andSouth America.

Dengue fever and yellow fever (YF) viruses belong to the same family offlaviviridae and share antigenic determinants, which may result incross-reacting antibodies. They are both transmitted between humans bymosquitoes (primarily Aedes aegypti), and are both endemic in tropicalareas of Africa and Latin America with a high public health impact.

Today there exists a yellow fever vaccine (YF-17D vaccine) which isbased on a live, attenuated viral strain, and is the only commerciallyavailable YF vaccine administered as a single subcutaneous injection.The YF-17D vaccine is highly effective (approaching 100%) and generallysafe with the exception of very rare cases of vaccine-associatedneurotropic and viscerotropic disease. Vaccination against YF is alsorequired for travelers to certain countries in accordance with theInternational Health Regulations, and is also recommended by the WHO forall subjects travelling to areas where there is evidence of persistentor periodic YF virus transmission. A YF-17D vaccine is available underthe product name YF-VAX® from Sanofi.

A yellow fever vaccine is recommended for people from nine months of ageand older who are living in or traveling to endemic areas, personstravelling to or through countries requiring. A single dose of yellowfever vaccine administered subcutaneously is usually sufficient toconfer sustained lifelong protective immunity against yellow fever.However, for people who remain at risk, a booster dose is recommendedevery 10 years.

Hence, there is a need for a safe and effective method of simultaneouslypreventing dengue disease and yellow fever.

Hepatitis A is a liver disease caused by the hepatitis A virus (HAV).The virus is primarily spread when an uninfected (and unvaccinated)person ingests food or water that is contaminated with the feces of aninfected person. The disease is closely associated with unsafe water orfood, inadequate sanitation and poor personal hygiene. The virus canalso be transmitted through close physical contact with an infectiousperson. Unlike hepatitis B and C, hepatitis A infection does not causechronic liver disease and is rarely fatal, but it can cause debilitatingsymptoms and fulminant hepatitis (acute liver failure), which is oftenfatal. Hepatitis A occurs sporadically and in epidemics worldwide, witha tendency for cyclic recurrences.

The hepatitis A virus is one of the most frequent causes of foodborneinfection. Epidemics related to contaminated food or water can eruptexplosively, such as the epidemic in Shanghai in 1988 that affectedabout 300,000 people. Hepatitis A viruses persist in the environment andcan withstand food-production processes routinely used to inactivateand/or control bacterial pathogens. The disease can lead to significanteconomic and social consequences in communities. It can take weeks ormonths for people recovering from the illness to return to work, school,or daily life. The impact on food establishments identified with thevirus, and local productivity in general, can be substantial. Indeveloping countries with poor sanitary conditions and hygienicpractices, most children (90%) have been infected with the hepatitis Avirus before the age of 10 years.

The number of people traveling internationally has grown substantiallyin recent decades. According to the United Nations World TourismOrganization (UNWTO), over 1.1 billion tourists travelled abroad in2014. The risk of becoming ill during international travel depends onmany factors, such as the region of the world visited, the length of thetrip, and the diversity of planned activities. Vaccine recommendationsare a prominent part of health preparations before international travel.Vaccination against hepatitis A virus is commonly recommended fortravelers to at-risk areas around the world including Asia, Africa, andLatin America.

For routine hepatitis A vaccination, a two-dose schedule is recommended,particularly in travelers at substantial risk of contracting hepatitis Aand in immunocompromised individuals. However, in healthy individuals,comparable effectiveness has been achieved with a single dose. Thevaccination schedule for children/adolescents (12 months through 18years of age) as well as for adults (≥19 years of age) consists of aprimary dose administered intramuscularly, and a further booster doseadministered intramuscularly 6 to 18 months later.

Available hepatitis A vaccines include HAVRIX® and VAQTA®.

Hence, there is a need for a safe and effective method of simultaneouslypreventing dengue disease and hepatitis A.

Human papillomavirus (HPV) is a common virus that is passed from oneperson to another through direct skin-to-skin contact during sexualactivity. Most sexually active people will be infected with HPV at sometime in their lives and the infection is most common in people in theirlate teens and early 20s. There are about 40 types of HPV that caninfect the genital areas of men and women. While most HPV types cause nosymptoms and are cleared by the body's immune system, some HPV types arepersistent and can cause cervical cancer in women and other less commoncancers—like cancers of the anus, penis, vagina, vulva and oropharynx orwarts in the genital areas of men and women, called genital warts.

HPV vaccination prevents HPV-associated cervical cancers as well asHPV-associated cancers of the anus, vulva, vagina, and oropharynx. Thevaccination can also prevent HPV-associated genital warts. HPVvaccination is recommended in particular for 11 and 12 year-old girls.It is also recommended for girls and women age 13 through 26 years ofage who have not yet been vaccinated or completed the vaccine series.HPV vaccine can also be given to girls beginning at age 9 years. The CDCrecommends 11 to 12 year olds girls get two doses of HPV vaccine toprotect against cancers caused by HPV. More recently, vaccination ofboys in the same age ranges has also been recommended.

The routine HPV vaccination schedule for adolescents who start thevaccination series before the 15th birthday includes two doses of a HPVvaccine. The two doses are usually separated by 6 to 12 months. Theminimum interval between doses is five calendar months. A three doseschedule is recommended for subjects who start the series on or afterthe 15th birthday and for subjects with certain immunocompromisingconditions (such as cancer, HIV infection, or taking immunosuppressivedrugs). The second dose is usually given 1 to 2 months after the firstdose and the third dose 6 months after the first dose. The minimuminterval between the first and second doses of vaccine is usually 4weeks. The minimum interval between the second and third doses ofvaccine is usually 12 weeks. The minimum interval between the first andthird doses is usually 5 calendar months. If the vaccination series isinterrupted, the series does not need to be restarted.

Available HPV vaccines include Gardasil® 9, which is a recombinant9-valent HPV (9vHPV) vaccine for preventing HPV serotypes 6, 11, 16, 18,31, 33, 45, 52, and 58. The HPV vaccine does not include any live orinactivated HPV, but the L1 proteins of the respective HPV serotypes.

Hence, there is a need for a safe and effective method of simultaneouslypreventing dengue disease and HPV-associated cancers or genital warts.

Measles is a highly contagious infectious disease caused by the measlesvirus, a single-stranded, negative-sense, enveloped (non-segmented) RNAvirus of the genus Morbillivirus within the family Paramyxoviridae.Complications occur in about 30% of cases and may include diarrhea,blindness, inflammation of the brain, and pneumonia, among others.Encephalitis occurs in approximately one of every 2000 reported cases;survivors often have permanent brain damage and mental retardation.Death, predominantly from respiratory and neurological causes, occurs inone of every 3000 reported measles cases. The risk of death is greaterfor infants and adults than for children and adolescents. Contractingmeasles during pregnancy increases fetal risk. Most commonly, this riskinvolves premature labor and moderately increases rates of spontaneousabortion and of low birth weight. Subacute sclerosing panencephalitis, aslow virus infection of the central nervous system, is associated withmeasles virus. Measles is an airborne disease which spreads easilythrough the coughs and sneezes of infected people and may also be spreadthrough contact with saliva or nasal secretions.

Mumps is an acute disease of children and young adults, caused by themumps virus, a single-stranded, negative-sense RNA virus of the genusRubulavirus within the family Paramyxoviridae. Mumps virus produces nosymptoms in about one-third of infected people. In those with a clinicalresponse, glandular and nerve tissue are most often affected and themost common symptoms include fever and swelling of the parotid glands.Complications may include meningitis (15%), pancreatitis (4%),inflammation of the heart, or permanent deafness. Frequent viruria andabnormal renal function suggest that mumps virus may infect the kidneys.Mumps is highly contagious and spreads rapidly among people livingclosely together by respiratory droplets or direct contact with aninfected person.

Rubella (German measles) is an infection caused by the rubella virus, asingle-stranded, positive-sense RNA virus of the genus Rubivirus withinthe family Togaviridae. The virus usually results in a mild illness,accompanied by few constitutional symptoms, and occurs most commonly inchildhood. If the infection occurs in a woman in early pregnancyhowever, the virus may cross the placenta to reach the fetus, in whichthe infection can induce birth defects. These defects may be serious andpermanent and include congenital heart disease, cataract formation,deafness and mental retardation. Rubella is usually spread through theair via coughs of people who are infected.

Combined vaccine for measles, mumps and rubella (MMR) is used widely forthe immunization of children in certain regions of the world, because ofits advantages over the individual vaccines. Combined vaccine provokesan adequate immune response in children simultaneously for the threeinfections.

MMR vaccines are indicated for simultaneous vaccination against measles,mumps, and rubella in individuals 12 months of age or older. Individualsfirst vaccinated at 12 months of age or older should be revaccinatedprior to elementary school entry. Revaccination is intended toseroconvert those who do not respond to the first dose. The AdvisoryCommittee on Immunization Practices (ACIP) recommends administration ofthe first dose at 12 to 15 months of age and administration of thesecond dose at 4 to 6 years of age.

Hence, there is a need for a safe and effective method of simultaneouslypreventing dengue disease and measles, mumps and rubella.

Tetanus is caused by an infection with the bacterium Clostridium tetaniwhich is commonly found in soil, saliva, dust, and manure. The bacteriagenerally enter the body through a break in the skin such as a cut orpuncture wound by a contaminated object. The bacteria produce toxinsthat interfere with muscle contractions, resulting in the typical signsof muscle spasms. It affects the brain and nervous system and causesextremely painful muscle spasms, usually all over the body. Spasms ofthe jaw can make it impossible to open the mouth, a condition called“lockjaw.” Tetanus kills one out of ten people infected with thedisease.

Diphtheria is an infection caused by the bacterium Corynebacteriumdiphtheriae which primarily infects the throat and upper airways, andproduces a toxin affecting other organs. Diphtheria has an acute onsetand the main characteristics are sore throat, low fever and swollenglands in the neck. The toxin may, in severe cases, cause myocarditis orperipheral neuropathy. The diphtheria toxin causes a membrane of deadtissue to build up over the throat and tonsils, making breathing andswallowing difficult. Diphtheria is a very contagious infection and thebacteria usually spread between people by direct contact or through theair, but it may also be spread by contaminated objects.

Pertussis, or whooping cough, caused by the bacterium Bordetellapertussis is an airborne disease that results in an extremely contagiousrespiratory infection that can lead to severe breathing problems,especially in infants. Pertussis first appears like an ordinary cold,but then causes intense, uncontrollable coughing spells which can causedifficulty breathing, vomiting, and disturbed sleep. A person may coughso hard that they vomit, break ribs, or become very tired from theeffort. Children less than one year old may have little or no cough andinstead have periods where they do not breathe. A high-pitched “whoop”noise is heard when the person tries to take a breath after coughing.Complications include pneumonia or death. Pertussis can affect people ofall ages, but can be very serious and even deadly, for babies less thana year old.

Tdap is a combination vaccine that protects against the threepotentially life-threatening bacterial diseases tetanus, diphtheria, andpertussis (whooping cough). Tdap stands for tetanus and diphtheriatoxoids with acellular pertussis. Tdap is an inactive vaccine producedby using dead bacteria. It is recommended that vaccination againsttetanus, diphtheria and pertussis carried out in infancy (in children ofless than 7 years of age) is done by using a particular diphtheriatoxoid, tetanus toxoid and acellular pertussis absorbed vaccine, such asINFANRIX® from GlaxoSmithKline. A further booster Tdap vaccine isrecommended for children of greater than 10 years to ensure thatimmunity against tetanus, diphtheria and pertussis is maintained intoadulthood. A booster Tdap vaccine based on combined tetanus toxoid,reduced diphtheria toxoid and acellular pertussis (adsorbed) isavailable under the brand name BOOSTRIX® from GlaxoSmithKline. Tdapvaccination is administered intramuscularly as a single dose.

Hence, there is a need for a safe and effective method of simultaneouslypreventing dengue disease and tetanus, diphtheria, and pertussis.

In addition to diphtheria, tetanus and pertussis, poliomyelitis, oftencalled polio or infantile paralysis, is a highly infectious viraldisease caused by the poliovirus. It invades the nervous system, and cancause total paralysis in a matter of hours. The virus is transmitted byperson-to-person spread mainly through the fecal-oral route or, lessfrequently, by a common vehicle (for example, contaminated water orfood) and multiplies in the intestine. Initial symptoms are fever,fatigue, headache, vomiting, stiffness of the neck and pain in thelimbs. 1 in 200 infections leads to irreversible paralysis (usually inthe legs). Among those paralyzed, 5% to 10% die when their breathingmuscles become immobilized. Polio mainly affects children under 5 yearsof age. There is no cure for polio, it can only be prevented.

Haemophilus influenzae type b (Hib) is a bacteria responsible for severepneumonia, meningitis and other invasive diseases almost exclusively inchildren aged less than 5 years. It is transmitted through therespiratory tract from infected to susceptible individuals. Hib alsocauses potentially severe inflammatory infections of the face, mouth,blood, epiglottis, joints, heart, bones, peritoneum, and trachea.Although this problem occurs worldwide the burden of Hib disease wasconsiderably higher in resource-poor countries, prior to theintroduction of the vaccine into their national immunization programs.In 2000, Hib was estimated to have caused two to three million cases ofserious disease, notably pneumonia and meningitis, and 386,000 deaths inyoung children. Hib disease is observed in all parts of the world but isdifficult to confirm because it requires prompt laboratory investigationin patients that have not received prior antibiotic treatment.

Commercially available combined DTaP/IPV/Hib vaccines include Pentacel®,which is based on combined diphtheria toxoid, tetanus toxoid, acellularpertussis (adsorbed), and inactivated poliovirus in combination with aHib conjugate vaccine.

Hence, there is a need for a safe and effective method of simultaneouslypreventing dengue disease and diphtheria, tetanus, pertussis,poliomyelitis and diseases caused by Haemophilus influenzae type b.

Furthermore, it has been reported that elderly subjects infected withdengue virus often present atypically, wherein fever may be the onlysymptom and are less likely to present with typical symptoms and signsof dengue fever which may be defined as fever plus at least one of thefollowing symptoms: bone pain, myalgia, arthralgia, retro-orbital pain,headache and maculopapular rash (Lee et al. (2013) Am. J. Emerg. Med.31(5): 783-787). Yet, elderly adults are at increased risk of developingsevere dengue and DHF/DSS compared to younger subjects (Liu et al.(2008) Am. J. Infect. Dis. 4(1): 10-17; Lin et al. (2012) Emerg. Infect.Dis. 18(10): 2003-1009; Rowe et al. (2014) PloS Negl. Trop. Dis. 8(4)).Several reasons which may contribute to severe disease in the elderlyhave been discussed, such as impaired physiological and immunefunctions, an increased probability of acquiring secondary dengue and anincreased prevalence of chronic diseases and other comorbidities in theelderly, i.e. above 60 (see the discussion in Lin et al. (2017) ExpertReview of Anti-infective Therapy 15(8): 729-735).

OBJECTS AND SUMMARY

It is an object of the present invention to provide a safe and effectivevaccine and corresponding method of inoculation against all serotypes ofdengue virus for dengue-endemic and dengue non-endemic populations andfor a broad range of ages, in particular for subjects between 2 monthsand 60 years of age, and independent of previous exposure to denguevirus and corresponding seropositive or seronegative status beforevaccination.

It is an object of the present invention to minimize the risk of DHF andDSS caused by infection with DENV-1, DENV-2, DENV-3 or DENV-4, inparticular following vaccination in children of young age andindividuals of any age who have never been previously exposed to dengue,or who are seronegative to dengue before vaccination.

It is an object of the invention to provide a vaccine and correspondingmethod of inoculating for controlling a dengue outbreak situation.

It is an object of the invention to provide a vaccine and correspondingmethod of inoculating which is useful in settings less familiar withclinical management of dengue or in those with fewer resources.

It is an object of the invention to provide a vaccine and correspondingmethod of inoculating which avoids testing for individual serostatusbefore individual inoculation or analysis of seroprevalence rates inareas to be mass vaccinated.

It is an object of the invention to provide a vaccine and correspondingmethod of inoculating which reduces and/or avoids the risk for laterantibody dependent enhancement.

It is an object of the invention to provide a vaccine and correspondingmethod which reduces and/or avoids dengue NS1 toxicosis.

It is an object of the invention to provide a vaccine and correspondingmethod of inoculating which induces cross reactive multitypic antibodyand/or cell mediated immunity.

It is one object of the present invention to provide a safe andeffective protection against dengue disease and yellow fever.

It is another object of the present invention to provide a safe andeffective protection against dengue disease and hepatitis A.

It is a further object of the present invention to provide a safe andeffective protection against dengue disease and HPV-associated cancersor genital warts.

It is a certain object of the present invention to provide a safe andeffective protection against dengue disease and measles, mumps andrubella.

It is another object of the present invention to provide safe andeffective protection against dengue disease and tetanus, diphtheria, andpertussis.

It is another object of the present invention to provide a safe andeffective protection against dengue disease and diphtheria, tetanus,pertussis, poliomyelitis and diseases caused by Haemophilus influenzaetype b.

It is another object of the present invention to provide safe andeffective protection against any combination of the above mentioneddiseases.

It is another object of the present invention to provide a vaccine whichstimulates a balanced immune response to all four dengue serotypes in asubject, in particular in an elderly subject (i.e. more than 60 years ofage).

The present invention is therefore directed in part to a dengue vaccinefor a method of mass vaccination without individual testing forserostatus or prior evaluation of seroprevalence rates. This massvaccination includes endemic regions including outbreak situations aswell as in non-endemic regions for travelers.

The present invention is therefore directed to a tetravalent denguevirus composition including four live attenuated dengue virus strainsrepresenting serotype 1, serotype 2, serotype 3 and serotype 4, whereinpreferably the tetravalent dengue virus composition includes a chimericdengue serotype 2/1 strain and a dengue serotype 2 strain and a chimericdengue serotype 2/3 strain and a chimeric dengue serotype 2/4 strain,wherein preferably the dengue serotype 2 strain is derived from the wildtype virus strain DEN-2 16681 (represented by SEQ ID NO 11) and differsin at least three nucleotides from the wild type as follows:

a) 5′-noncoding region (NCR)-57 (nt-57 C-to-T)

b) NS1-53 Gly-to-Asp (nt-2579 G-to-A)

c) NS3-250 Glu-to-Val (nt-5270 A-to-T); and

the three chimeric dengue strains being derived from the serotype 2strain by replacing the structural proteins prM and E from serotype 2strain with the corresponding structural proteins from the other dengueserotypes, resulting in the following chimeric dengue strains:

a DENV-2/1 chimera,

a DENV-2/3 chimera and

a DENV-2/4 chimera.

The present invention is in particular directed to a composition orlyophilized unit dose which upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent comprises:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The present invention is also in particular directed to such acomposition or unit dose which upon reconstitution with apharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide atotal concentration of pfu/0.5 mL and based on said total concentrationthe concentration of (ii) in pfu/0.5 mL is less than 10%, and theconcentration of (iv) in pfu/0.5 mL is at least 50%, and theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 8%, or preferably at least 10%, or atleast 12%, or at least 14%, or at least 16%, or at least 18%.

The present invention is also in particular directed to such method anduse wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is less than 2%, the concentration of (iv) in pfu/0.5mL is at least 50%, the concentration of (i) in pfu/0.5 mL is at least1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%.

The present invention is therefore directed to a method including andcorresponding use of a tetravalent dengue virus composition includingfour live attenuated dengue virus strains representing serotype 1,serotype 2, serotype 3 and serotype 4, the method being directed toinoculating a subject against virologically confirmable dengue disease,wherein in particular the tetravalent dengue virus composition includesa chimeric dengue serotype 2/1 strain and a dengue serotype 2 strain anda chimeric dengue serotype 2/3 strain and a chimeric dengue serotype 2/4strain, wherein in particular the dengue serotype 2 strain is derivedfrom the wild type virus strain DEN-2 16681 (represented by SEQ ID NO11) and differs in at least three nucleotides from the wild type asfollows:

d) 5′-noncoding region (NCR)-57 (nt-57 C-to-T)

e) NS1-53 Gly-to-Asp (nt-2579 G-to-A)

f) NS3-250 Glu-to-Val (nt-5270 A-to-T); and

the three chimeric dengue strains being derived from the serotype 2strain by replacing the structural proteins prM and E from serotype 2strain with the corresponding structural proteins from the other dengueserotypes, resulting in the following chimeric dengue strains:

a DENV-2/1 chimera,

a DENV-2/3 chimera and

a DENV-2/4 chimera.

The present invention is in particular directed to such method and usewherein the unit dose is lyophilized and upon reconstitution with 0.5 mLof a pharmaceutically acceptable diluent comprises:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The present invention is also in particular directed to such method anduse wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) inpfu/0.5 mL is at least 50%, and the concentration of (i) in pfu/0.5 mLis at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%,or at least 18%, and wherein the subject is preferably 2 to 17 years ofage or 4 to 16 years of age.

The present invention is also in particular directed to such method anduse wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is less than 2%, the concentration of (iv) in pfu/0.5mL is at least 50%, the concentration of (i) in pfu/0.5 mL is at least1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%, whereinthe subject is 18 to 60 years of age.

The present invention is therefore directed to a method andcorresponding use, the method comprising a primary vaccination with onlytwo administrations of the unit dose comprising the steps of:

-   (A) administering a first unit dose of the tetravalent dengue virus    composition to the subject, and-   (B) administering a second unit does of the tetravalent dengue virus    composition to the subject within 3 months of administration of the    first unit dose,    wherein preferably the method and use do not include a step of    determination whether there was a previous dengue infection in the    subject before administration of the unit dose or wherein the    serostatus of the subject is unknown before administration of the    unit dose or wherein the method and use do not include a step of    determination of a previous dengue infection in the subjects    preferably at any time before, during or after the steps of    administration or wherein the serostatus of the subject is unknown    preferably at any time before, during or after the steps of    administration.

According to one embodiment such a method and use do not include theactive surveillance with respect to febrile illness of the subject afterthe administration of the first- and second-unit dose. During activesurveillance any subject with febrile illness (defined as fever ≥38° C.on any 2 of 3 consecutive days) will be asked to return to the site fordengue fever evaluation by the Investigator. Subjects/guardians will becontacted at least weekly to ensure robust identification of febrileillness by reminding subjects/guardians of their obligation to return tothe site in case of febrile illness. This contact will be implementedthrough appropriate methods that may differ in each trial site (eg,phone calls, text messaging, home visits, school-based surveillance).

According to one embodiment such a method and use do not include vaccineimmunogenicity analysis including GMTs for dengue neutralizingantibodies.

According to one embodiment such a method and use do not include areactogenicity analysis. Such a reactogenicity analysis relates tosolicited local AEs (injection site pain, injection site erythema, andinjection site swelling) and solicited systemic AEs (child <6 years:fever, irritability/fussiness, drowsiness and loss of appetite; child ≥6years: asthenia, fever, headache, malaise and myalgia) which will e.g.be assessed for 7 days and 14 days, respectively, following eachvaccination (vaccination day included) via collection of diary cards.

The method according to the invention does not require the testing ofthe serostatus before vaccination and thus allows immediate treatmentand outbreak control. According to certain embodiments the invention isdirected to a method and use wherein the subject is exposed to a dengueoutbreak. In certain such embodiments the outbreak is due to a dengueserotype 2, and/or due to a serotype 1.

According to certain embodiments the method of inoculation against thevirologically confirmable dengue disease is due to a dengue serotype 2,and/or due to a dengue serotype 1.

According to one embodiment the method comprises a primary vaccinationconsisting of the steps of:

(A) selecting a subject for administration of the unit doses of thetetravalent dengue virus composition in need for protection againstdengue infection without determination of a previous dengue infection,and(B) administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and(C) administering a second unit dose of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.Therefore the method of inoculating is finalized without determinationof a previous dengue infection. The method further optionally comprisesat least 1 years after the administration of the second unit dose abooster dose of the unit dose.

According to one embodiment the method and use is directed to a methodof inoculating a subject against virologically confirmable denguedisease with a tetravalent dengue virus composition including four liveattenuated dengue virus strains representing serotype 1, serotype 2,serotype 3 and serotype 4, the dengue serotype 2 strain being derivedfrom the wild type virus strain DEN-2 16681 (represented by SEQ ID NO11) and differing in at least three nucleotides from the wild type asfollows:

a) 5′-noncoding region (NCR)-57 (nt-57 C-to-T)b) NS1-53 Gly-to-Asp (nt-2579 G-to-A)c) NS3-250 Glu-to-Val (nt-5270 A-to-T); andthe three chimeric dengue strains being derived from the serotype 2strain by replacing the structural proteins prM and E from serotype 2strain with the corresponding structural proteins from the other dengueserotypes, resulting in the following chimeric dengue strains:

a DENV-2/1 chimera,

a DENV-2/3 chimera and

a DENV-2/4 chimera,

the method comprises a primary vaccination consisting of the steps of:

-   (A) selecting a subject for administration of the unit doses of the    tetravalent dengue virus composition in need for protection against    dengue infection without determination whether there was a previous    dengue infection, and-   (B) administering a first unit dose of the tetravalent dengue virus    composition to the subject, and-   (C) administering a second unit dose of the tetravalent dengue virus    composition to the subject within 3 months of administration of the    first unit dose.    Therefore the method of inoculating is finalized without    determination of a previous dengue infection. The method further    optionally comprises at least 1 years after the administration of    the second unit dose a booster dose of the unit dose.

According to the invention the method and use is applicable to subjectsof all kinds of ages. The present invention is in particular directed tosuch a method and use wherein the subject is under 9 years of age, or 4to 5 years of age, or 6 to 11 years of age, or 12 to 16 years, or 6 to16 years of age, or 4 to 16 years of age, or 2 to 17 years of age, or 9years of age, or over 9 years of age, or 9 to 17 years of age, or 18 to60 years of age, or 18 to 45 years of age, or 46 to 60 years of age, orover 60 years of age.

In particular the present invention is directed to such use wherein themethod is safe.

In particular the present invention is directed to such use wherein themethod provides a combined vaccine efficacy againstvirologically-confirmed dengue with hospitalization against all fourserotypes with a 2-sided 95% confidence interval, wherein the lowerbound is more than 65%, when measured against placebo in a subjectpopulation of at least 5,000 healthy 4 to 16 year old subjectsirrespective of serostatus at baseline from first administration of theadministration schedule until 12 to 18 months after the lastadministration of the administration schedule.

In particular the present invention is directed to such use wherein themethod is effective.

In particular the present invention is directed to such use wherein themethod provides a combined vaccine efficacy against all four serotypes,in preventing virologically confirmable dengue disease with a 2-sided95% confidence interval, wherein the lower bound is more than 60%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects irrespective of serostatus at baseline and 14 to 16years of age, from the first administration of the administrationschedule until 18 months after the last administration of theadministration schedule.

The present invention is also directed in part to a reconstituted denguevaccine composition for use in a method of preventing virologicallyconfirmable dengue disease in a subject comprising consecutivelyadministering at least a first and a second unit dose of the denguevaccine composition to the subject, wherein said first and second unitdose are administered subcutaneously within 3 months and at least 4weeks apart, optionally at about day 1 and at about day 90, wherein thedengue vaccine composition is a tetravalent dengue virus compositionincluding four dengue virus strains representing dengue serotype 1,dengue serotype 2, dengue serotype 3 and dengue serotype 4, optionallywherein the dengue virus strains are live, attenuated, and wherein uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluent

-   -   (i) dengue serotype 1 has a concentration of at least 3.3 log 10        pfu/0.5 mL and optionally to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of at least 2.7 log        10 pfu/0.5 mL and optionally to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of at least 4.0 log        10 pfu/0.5 mL and optionally to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of at least 4.5 log        10 pfu/0.5 mL and optionally to 6.2 log 10 pfu/0.5 mL.

The present invention is therefore directed in part to a dengue vaccinecomposition for use in a method of preventing virologically confirmabledengue disease (VCD) in a subject comprising consecutively administeringat least a first and a second unit dose of the dengue vaccinecomposition to the subject, wherein said first and second unit dose areadministered subcutaneously within 3 months and at least 4 weeks apart,optionally at about day 1 and at about day 90, and wherein the denguevaccine composition is a tetravalent dengue virus composition includingfour live, attenuated dengue virus strains representing dengue serotype1, dengue serotype 2, dengue serotype 3 and dengue serotype 4, whereinthe attenuated dengue virus strains comprise chimeric dengue viruses andpreferably at least one non-chimeric dengue virus, and wherein thedengue serotype 1 and the dengue serotype 2 are present each in aconcentration based on the total concentration in pfu/0.5 mL which iswithin 5%-points of each other and/or are together less than about 10%of the total concentration in pfu/0.5 mL, in particular wherein thedengue serotype 3 is at least about 10% of the total concentration inpfu/0.5 mL and in particular wherein the dengue serotype 4 is at leastabout 70% of the total concentration in pfu/0.5 mL.

The present invention is therefore directed in part to a unit dose of adengue vaccine composition and use thereof, the unit dose comprising:

a tetravalent dengue virus composition including four live attenuateddengue serotypes (e.g. virus strains):

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain),    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain),    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain),    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain),        and one or more pharmaceutically acceptable excipients thereof.

The present invention is further directed in part to a unit dose of adengue vaccine composition and use thereof, the unit dose comprising:

a tetravalent virus composition including four live attenuated denguevirus strains:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) in a concentration of at least 3.3 log 10 pfu/0.5 ml,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) in a        concentration of at least 2.7 log 10 pfu/0.5 ml,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) in a concentration of at least 4.0 log 10 pfu/0.5 ml,        and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) in a concentration of at least 4.5 log 10 pfu/0.5 ml,        and one or more pharmaceutically acceptable excipients.

The present invention is further directed in part to a unit dose of adengue vaccine composition and use thereof, the unit dose comprising:

a tetravalent virus composition including four live attenuated denguevirus strains, wherein the unit dose is lyophilized and uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) in a concentration of at least 3.3 log 10 pfu/0.5 ml,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) in a        concentration of at least 2.7 log 10 pfu/0.5 ml,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) in a concentration of at least 4.0 log 10 pfu/0.5 ml,        and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) in a concentration of at least 4.5 log 10 pfu/0.5 ml.

The present invention is further directed in part to a unit dose of adengue vaccine composition and use thereof, wherein said unit dose islyophilized and obtained by lyophilizing 0.5 mL of a solutioncomprising:

a tetravalent virus composition including four live attenuated denguevirus strains:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) in a concentration of at least 3.3 log 10 pfu/0.5 ml,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) in a        concentration of at least 2.7 log 10 pfu/0.5 ml,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) in a concentration of at least 4.0 log 10 pfu/0.5 ml,        and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) in a concentration of at least 4.5 log 10 pfu/0.5 ml,        and one or more pharmaceutically acceptable excipients.

The present invention is in particular directed to such unit dose orcomposition wherein upon reconstitution with a pharmaceuticallyacceptable diluent (i), (ii), (iii), and (iv) provide a totalconcentration of pfu/0.5 mL and based on said total concentration theconcentration of (ii) in pfu/0.5 mL is less than 10%, and theconcentration of (iv) in pfu/0.5 mL is at least 50%, and theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 8%, or at least 10%, or at least 12%,or at least 14%, or at least 16%, or at least 18%, in particular whereinthe concentration of (iii) in pfu/0.5 mL is at least 10%.

The present invention is in particular directed to such unit dose orcomposition, wherein upon reconstitution with a pharmaceuticallyacceptable diluent (i), (ii), (iii), and (iv) provide a totalconcentration of pfu/0.5 mL and based on said total concentration theconcentration of (ii) in pfu/0.5 mL is less than 2%, the concentrationof (iv) in pfu/0.5 mL is at least 50%, the concentration of (i) inpfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mLis at least 6%.

The present invention is further directed in part to a kit for preparinga reconstituted unit dose and use thereof, the kit comprising thefollowing components:

a) a lyophilized unit dose of the present invention as described herein,andb) a pharmaceutically acceptable diluent for reconstitution.

The present invention is further directed in part to a container, suchas a vial, comprising one to ten unit doses of the present invention asdescribed herein.

The present invention is further directed to a method of preventingdengue disease in a subject comprising administering to the subject areconstituted unit dose of a dengue vaccine composition as describedherein, for example by subcutaneous injection.

The present invention is further directed to the use of thereconstituted unit dose of a dengue vaccine composition as describedherein for the manufacture of a medicament for preventing dengue diseasein a subject, for example by subcutaneous injection.

The present invention is further directed to the reconstituted unit doseof a dengue vaccine composition as described herein for use in a methodof preventing dengue disease in a subject, for example by subcutaneousinjection.

The present invention is further directed to a method of preventingdengue disease in a subject population, comprising administering to thesubject population a reconstituted unit dose of a vaccine composition asdescribed herein, wherein the subject population is seronegative to alldengue serotypes. In said method the geometric mean neutralizingantibody titers (GMTs) when tested in at least 40, or at least 50, or atleast 60 subjects at day 180 or day 365 after at least a firstadministration of said unit dose, and optionally a second administrationof said unit dose 90 days after said first administration, may provide aratio of not more than 50, or not more than 40, or nor more than 30, ornot more than 20 for the GMT of dengue serotype 2 to the GMT of dengueserotype 4 (GMT DENV-2:GMT DENV-4), and optionally a ratio of not morethan 20 for the GMT of dengue serotype 2 to the GMT of dengue serotype 1(GMT DENV-2:GMT DENV-1), and optionally a ratio of not more than 20 forthe GMT of dengue serotype 2 to the GMT of dengue serotype 3 (GMTDENV-2:GMT DENV-3).

The present invention is further directed to a method of preventingdengue disease in a subject, comprising administering to the subject areconstituted unit dose of a vaccine composition as described herein,wherein the subject is seronegative to all dengue serotypes. In saidmethod the neutralizing antibody titers when tested in the subject atday 180 or day 365 after at least a first administration of said unitdose, and optionally a second administration of said unit dose 90 daysafter said first administration, may provide a ratio of not more than50, or not more than 40, or nor more than 30, or not more than 20 forthe neutralizing antibody titer of dengue serotype 2 to the neutralizingantibody titer of dengue serotype 4, and optionally a ratio of not morethan 20 for the neutralizing antibody titer of dengue serotype 2 to theneutralizing antibody titer of dengue serotype 1, and optionally a ratioof not more than 20 for the neutralizing antibody titer of dengueserotype 2 to the neutralizing antibody titer of dengue serotype 3.

In one preferred embodiment, the methods of preventing dengue disease ofthe present invention are not associated with an increased likelihood ofsolicited systemic adverse events, such as in children under 9 orseronegative individuals.

The present invention is therefore directed in part to a method ofpreventing dengue disease as well as yellow fever.

The present invention is therefore directed in part to a method ofpreventing dengue disease as well as hepatitis A.

The present invention is therefore directed in part to a method ofpreventing dengue disease as well as

HPV-associated cancers or genital warts.

The present invention is therefore directed in part to a method ofpreventing dengue disease as well as measles, mumps and rubella.

The present invention is therefore directed in part to a method ofpreventing dengue disease as well as tetanus, diphtheria, and pertussis.

The present invention is therefore directed in part to a method ofpreventing dengue disease as well as diphtheria, tetanus, pertussis,poliomyelitis and diseases caused by Haemophilus influenzae type b.

The present invention is therefore also directed in part to a method ofprevention any combination of the above mentioned diseases.

The present invention is further directed in part to the use of thereconstituted unit dose of a dengue vaccine composition/tetravalentdengue virus composition as described herein for the manufacture of amedicament for preventing dengue disease in a subject, for example bysubcutaneous injection.

The present invention is further directed in part to the reconstitutedunit dose of a dengue vaccine composition/tetravalent dengue viruscomposition as described herein for use in a method of preventing denguedisease in a subject, as described herein.

Definitions

In describing the present invention, the following terms are to be usedas indicated below. As used herein, the singular forms “a,” “an,” and“the” include plural references unless the context clearly indicatesotherwise.

As used herein, the terms “unit dose of a dengue vaccine composition”,“unit dose” and “unit dose of the invention as described herein” referto the amount of a dengue vaccine which is administered to a subject ina single dose. In one embodiment, one unit dose is present in a vial andthis unit dose is administered to a subject, e.g. optionally afterreconstitution. In one embodiment, more than one unit dose of the denguevaccine composition may be present in a vial so that with the content ofone vial more than one subject can be vaccinated.

A “lyophilized unit dose” or “unit dose in lyophilized form” refers tothe unit dose that is obtained by subjecting a given volume of theliquid dengue vaccine composition, such as 0.5 mL, to lyophilization.Thus, the aqueous formulations of the dengue vaccine composition beingproduced by combining the pharmaceutically acceptable excipients and thedengue virus composition comprising the four dengue virus strains,preferably TDV-1 to TDV-4, is subjected to lyophilization to obtain thelyophilized unit dose.

A “reconstituted unit dose” or “unit dose in reconstituted form” isobtained from the lyophilized dose by reconstitution with apharmaceutically acceptable diluent. The diluent does not contain denguevirus. The reconstituted unit dose is a liquid which can be administeredto a subject, for example by injection, such as subcutaneous injection.

As used herein, the term “upon reconstitution with 0.5 mL” is notlimiting the reconstitution to be performed using 0.5 mL of the diluent,but refers to the concentration of the dengue viruses that will bepresent in the reconstituted unit dose when 0.5 mL diluent are used forreconstitution. While using a different volume for reconstitution (e.g.0.8 mL) will result in a different concentration of dengue viruses inthe reconstituted unit dose, the administration of the total volume ofthe unit dose (e.g. 0.8 mL) will result in the same total amount ofdengue virus being administered.

As used herein, a “concentration of at least X log 10 pfu/0.5 mL” refersto the concentration of a dengue serotype in 0.5 mL, but is not limitingthe unit dose to be 0.5 mL. If the unit dose has a volume different than0.5 mL, or is lyophilized from a volume different than 0.5 mL, or isreconstituted with a volume different than 0.5 mL, said concentrationwill differ from the “concentration of at least X log 10 pfu/0.5 mL”.However, if the unit dose has a volume of 0.5 mL, or is lyophilized froma volume of 0.5 mL, or is reconstituted with a volume of 0.5 mL, saidconcentration will be the “concentration of at least X log 10 pfu/0.5mL”. Thus, while the concentration may differ, the total amount of virusin the unit dose remains the same.

As used herein, the term “dengue serotype” refers to a species of denguevirus which is defined by its cell surface antigens and therefore can bedistinguished by serological methods known in the art. At present, fourserotypes of dengue virus are known, i.e. dengue serotype 1 (DENV-1),dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) and dengueserotype 4 (DENV-4).

As used herein, the term “tetravalent dengue virus composition” refersto a dengue virus composition comprising four different immunogeniccomponents from the four different dengue serotypes DENV-1, DENV-2,DENV-3 and DENV-4, preferably comprising four different live, attenuateddengue viruses, each representing one dengue serotype, and which aims tostimulate immune responses to all four dengue serotypes.

As used herein, the term “live attenuated dengue virus” refers to aviable dengue virus which is mutated to provide reduced virulence. Thelive attenuated dengue virus can be a dengue virus in which allcomponents are derived from the same dengue serotype or it can be achimeric dengue virus having parts from two or more dengue serotypes.

A “virus strain” and in particular a “dengue virus strain” is a geneticsubtype of a virus, in particular of a dengue virus, which ischaracterized by a specific nucleic acid sequence. A dengue serotype maycomprise different strains with different nucleic acid sequences whichhave the same cell surface antigens. A dengue virus strain can be adengue virus in which all components are derived from the same dengueserotype or it can be a chimeric dengue virus having parts from two ormore dengue serotypes.

As used herein, “TDV-2” refers to a molecularly characterized and cloneddengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53virus strain. The PDK-53 strain is described for example inBhamarapravati et al. (1987) Bulletin of the World Health Organization65(2): 189-195. In one embodiment, the TDV-2 strain served as a backbonefor the chimeric TDV-1, TDV-3 and TDV-4 strains into which parts fromthe TDV-1, TDV-3 and TDV-4 strains were introduced.

A “non-chimeric dengue virus” or “non-chimeric dengue serotype strain”or “non-chimeric dengue strain” comprises only parts from one dengueserotype. In particular, a non-chimeric dengue virus does not includeparts from a different flavivirus such as yellow fever virus, Zikavirus, West Nile virus, Japanese encephalitis virus, St. Louisencephalitis virus, tick-borne encephalitis virus. TDV-2 is an exampleof a non-chimeric dengue virus.

A “chimeric dengue virus” or “chimeric dengue serotype strain” or“chimeric dengue strain” comprises parts from at least two differentdengue serotypes. As used herein, the chimeric dengue virus does notinclude parts from a different flavivirus such as yellow fever virus,Zika virus, West Nile virus, Japanese encephalitis virus, St. Louisencephalitis virus, tick-borne encephalitis virus. In particular, thechimeric dengue virus described herein does not include parts from theyellow fever virus. As used herein, a “chimeric dengue serotype 2/1strain” or “DENV-2/1 chimera” or “TDV-1” refers to a dengue viruschimeric construct which comprises parts from both DENV-2 and DENV-1. Inparticular, in the chimeric dengue serotype 2/1 strain the prM and Eproteins from DENV-1 replace the prM and E proteins from DENV-2 asdetailed below. As used herein, a “chimeric dengue serotype 2/3 strain”or “DENV-2/3 chimera” or “TDV-3” refers to a dengue virus chimericconstruct which comprises parts from both DENV-2 and DENV-3. Inparticular, in the chimeric dengue serotype 2/3 strain the prM and Eproteins from DENV-3 replace the prM and E proteins from DENV-2 asdetailed below. As used herein, a “chimeric dengue serotype 2/4 strain”or “DENV-2/4 chimera” or “TDV-4” refers to a dengue virus chimericconstruct which comprises parts from both DENV-2 and DENV-4. Inparticular, in the chimeric dengue serotype 2/4 strain the prM and Eproteins from DENV-4 replace the prM and E proteins from DENV-2 asdetailed below.

As used herein, “TDV” refers to a tetravalent live attenuated denguevaccine that comprises a mixture of the four live attenuated denguevirus strains TDV-1, TDV-2, TDV-3 and TDV-4 expressing surface antigensfrom the four dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4,respectively. In one embodiment (e.g. also in the examples), TDV-1 hasthe nucleotide sequence according to SEQ ID No. 1 and/or the amino acidsequence according to SEQ ID No. 2. In one embodiment, TDV-2 has thenucleotide sequence according to SEQ ID No. 3 and/or the amino acidsequence according to SEQ ID No. 4. In one embodiment, TDV-3 has thenucleotide sequence according to SEQ ID No. 5 and/or the amino acidsequence according to SEQ ID No. 6. In one embodiment, TDV-4 has thenucleotide sequence according to SEQ ID No. 7 and/or the amino acidsequence according to SEQ ID No. 8.

As used herein, the term “dengue disease” refers to the disease which iscaused by infection with dengue virus. Symptoms of dengue diseaseinclude sudden high fever, headaches, joint and muscle pain, nausea,vomiting and skin rashes. The term dengue disease also includes the moresevere forms of dengue hemorrhagic fever (DHF) and dengue shock syndrome(DSS). Symptoms of DHF include increased vascular permeability,hypovolemia and abnormal blood clotting mechanisms. Subjects with DHFmay present with severe manifestations of plasma leakage and hemorrhage.When a subject with DHF experiences shock he or she will be categorizedas having DSS. Symptoms of DSS include bleeding that may appear as tinyspots of blood on the skin and larger patches of blood under the skin.Prolonged shock is the main factor associated with complicationsincluding massive gastrointestinal hemorrhage that can lead to death. Asused herein, DHF cases are defined as VCD cases meeting WHO 1997 DHFcriteria. In the context of preventing dengue disease in elderlysubjects, the term “preventing dengue disease” preferably includespreventing DHF and/or DSS. In the context of preventing dengue diseasein elderly subjects, the term “preventing dengue disease” preferablyincludes preventing severe end-organ manifestations of dengue such ashepatomegaly and acute renal failure.

As used herein, “preventing dengue disease” refers to preventing asubject from developing one or more symptoms of dengue disease becauseof an infection with a dengue virus. In particular, preventing denguedisease is achieved by vaccinating or inoculating a subject with adengue vaccine composition, such as the reconstituted unit dosedescribed herein. As used herein, the term “prophylactically treatingdengue disease” is equivalent to “preventing dengue disease”. In aparticular embodiment, preventing dengue disease includes preventing DHSand/or DSS.

As used herein, the terms “virologically-confirmed dengue disease”, “VCDcase”, or “VCD fever” refer to febrile illness or illness clinicallysuspected to be dengue disease with a positive serotype-specific reversetranscriptase polymerase chain reaction (RT-PCR). The term“virologically confirmable dengue” disease refers to a subject havingfebrile illness or illness clinically suspected to be dengue disease,wherein testing the subject, e.g. using RT-PCR, would confirm thepresence of at least one dengue serotype. Severe forms of VCD fever willbe identified as follows: Dengue Hemorrhagic Fever (DHF) was definedaccording to the WHO 1997 criteria. Severe dengue was defined through anassessment of an independent Dengue Case Adjudication Committee whichwill assess all hospitalized VCD cases (severe/non-severe) based oncriteria redefined in a charter. All non-hospitalized cases areconsidered non-severe.

As used herein, the term “febrile illness” is defined as temperature 38°C. on any 2 of 3 consecutive days.

As used herein, the terms “virologically-confirmed dengue disease withhospitalization”, is considered to be a surrogate for severe dengue andthe “incidence of virologically-confirmed dengue disease withhospitalization” is used as a safety parameter. As used herein, the“relative risk with respect to virologically-confirmed dengue diseasewith hospitalization” means the number of events of virologicallyconfirmed dengue disease with hospitalization divided by the number ofsubjects treated with the unit dose as disclosed herein over the numberof events of virologically confirmed dengue disease with hospitalizationdivided by the number of subjects treated with placebo. If the “relativerisk with respect to virologically-confirmed dengue disease withhospitalization” is 1 or lower the vaccine provides for the same or lessrisk for virologically-confirmed dengue disease with hospitalization asplacebo and is considered “safe”. In this context the risk ofvirologically-confirmed dengue disease with hospitalization may be also0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 orless, 0.3 or less, 0.2 or less, or 0.1 or less, in particular whendetermined from 30 days after a second administration until 12 monthsafter a second administration, in particular when determined in agegroups selected from the age group of 4 to 16 year old subjects, the agegroup of 4 to under 9 year old subjects, the age group of 2 to under 9year old subjects, the age group of 4 to 5 year old subjects, the agegroup of 6 to 11 year old subjects, and the age group of 12 to 16 yearold subjects.

As used herein, alternatively a vaccine is considered “safe” when thevaccine efficacy (VE) with respect to virologically-confirmed denguedisease with hospitalization is 0% or higher. This means that thevaccine provides for the same likelihood or less forvirologically-confirmed dengue disease with hospitalization as placebo.In particular considered “safe” is the combined vaccine efficacy againstvirologically-confirmed dengue with hospitalization against all fourserotypes with a 2-sided 95% confidence interval, wherein the lowerbound is more than 25%, in particular when measured against placebo in asubject population of at least 1,500 or at least 2,000 healthy subjects(in particular when measured in age groups selected in particular fromthe age group of 4 to 16 year old subjects, the age group of 4 to under9 year old subjects, the age group of 2 to under 9 year old subjects,the age group of 4 to 5 year old subjects, the age group of 6 to 11 yearold subjects, and the age group of 12 to 16 year old subjects) beingseronegative against all serotypes at baseline or being seropositiveagainst at least one serotype at baseline, in particular when said unitdose or said placebo is administered at least twice within less than 6months, such as within 3 months, about from first administration or from30 days after the second or last administration of the administrationschedule until at least 12 months, until 12 to 18 months, until 12months, or until 18 months after the second or last administration ofthe administration schedule. In particular, the lower bound may be morethan 30%, more than 40%, more than 50%, more than 60%, more than 65%,more than 66%, more than 67%, more than 68% more than 70%, or more than75%. In particular, the 2-sided 95% confidence interval of the combinedvaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes when comparing seropositiveand seronegative subjects provides for lower bounds of the 2-sidedconfidence interval which are within 10% points or within 15% points orwithin 20% points. In a particular embodiment “safe” means providing acombined vaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 65%, when measuredagainst placebo in a subject population of at least 5,000 healthy 4 to16 year old subjects irrespective of serostatus at baseline from firstadministration of the administration schedule until 12 to 18 monthsafter the last administration of the administration schedule.

If one of the criteria as defined above for the term “safe” isfulfilled, the vaccine is considered safe within the meaning of thisinvention. In this context, safe in particular refers to a vaccine thatis safe for all subjects irrespective of their serostatus at baseline.This means that the vaccine can be administered without the need todetermine the occurrence of a previous dengue infection in the subjectbefore administration. Preferably, the vaccine is safe as defined abovewith respect to all age groups starting from 4 years of age andpreferably irrespective of the serostatus, in particular from 4 years ofage to 60 years of age, or 4 years of age to 16 years of age. Relevantsubgroups in this context are under 9 years of age, from 2 years of ageto under 9 years of age, from 4 years of age to under 9 years of age, 4to 5 years of age, 6 to 11 years of age and 12 to 16 years of age or anyage group within 4 to 16 years of age. For further definitions of VEagainst virologically-confirmed dengue disease with hospitalizationreference is made to the disclosure below with respect to certainmethods of treatment.

As used herein, “vaccine efficacy” or “VE” measure the proportionatereduction in cases among vaccinated persons. Vaccine efficacy (VE) ismeasured by calculating the risk of disease among vaccinated andunvaccinated persons and determining the percentage reduction in risk ofdisease among vaccinated persons relative to unvaccinated persons. Thegreater the percentage reduction of illness in the vaccinated group, thegreater the vaccine efficacy. For example, a VE of 90% indicates a 90%reduction in disease occurrence among the vaccinated group, or a 90%reduction from the number of cases you would expect if they have notbeen vaccinated. The vaccine efficiency is calculated by the formula:100*(1−HR), wherein HR is the Hazard Ratio which is defined as theHazard rate of vaccine (λv) divided by the Hazard rate of placebo (λc),i.e. HR=λv/λc. λv denote the hazard rate for the subjects vaccinatedwith a tetravalent dengue vaccine composition as disclosed herein and λcdenote the hazard rate for unvaccinated subjects, i.e. subjectsreceiving placebo. The hazard rate ratio HR is estimated from a Coxproportional hazard model with study vaccine as a factor, adjusted forage, and stratified by region. As used herein the term“combined vaccineefficacy against all four serotypes” is defined as the vaccine efficacyin relation to the risk of dengue disease irrespective of the serotypebeing responsible for the virologically-confirmed dengue disease and thesubject baseline serostatus. A vaccine is considered “effective” in casethe combined vaccine efficacy is above 30%. In this context the combinedvaccine efficacy may be also 40% or more, 50% or more, 60% or more, 70%or more, 72% or more, or 80% or more, in particular when determined from30 days after a second administration until 12 months after a secondadministration or 18 months after a second vaccination, in particularwhen determined in age groups selected from the age group of 4 to 16year old subjects, the age group of 4 to under 9 year old subjects, theage group of 2 to under 9 year old subjects, the age group of 4 to 5year old subjects, the age group of 6 to 11 year old subjects, and theage group of 12 to 16 year old subjects. In this context, effective inparticular refers to a vaccine that is effective for all subjectsirrespective of their serostatus at baseline. Preferably, the vaccine iseffective with respect to all age groups starting from 4 years of ageand preferably irrespective of the serostatus, in particular from 4years of age to 60 years of age or from 4 years of age to 16 years ofage and irrespective of the serostatus. Relevant subgroups in thiscontext are under 9 years of age, from 2 years of age to under 9 yearsof age, from 4 years of age to under 9 years of age, 4 to 5 years ofage, 6 to 11 years of age and 12 to 16 years of age or any age groupwithin 4 to 16 years of age. In certain embodiments “effective” meansproviding a combined vaccine efficacy against all four serotypes, inpreventing virologically confirmable dengue disease with a 2-sided 95%confidence interval, wherein the lower bound is more than 60%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects irrespective of serostatus at baseline and 4 to 16years of age, from the first administration of the administrationschedule until 18 months after the last administration of theadministration schedule. Further specific efficacies can be defined. Asused herein, “combined vaccine efficacy against all four serotypes inseronegative subjects” refers to the efficacy measured in subjects whichare seronegative at baseline. As used herein, “vaccine efficacy againsta specific serotype, e.g. serotype 1” refers to the efficacy in relationto a specific serotype being responsible for the virologically-confirmeddengue disease. As used herein, “combined vaccine efficacy against allfour serotypes against virologically-confirmed dengue withhospitalization” refers to the efficacy wherein onlyvirologically-confirmed dengue cases with hospitalization areconsidered. Such vaccine efficacies can be determined with respect tosubjects being seronegative or seropositive at baseline and fordifferent age groups.

As used herein, the “relative risk” means the number of events ofvirologically confirmed dengue disease divided by the number of subjectstreated with the unit dose as disclosed herein over the number of eventsof virologically confirmed dengue disease divided by the number ofsubjects treated with placebo. As used herein the term“combined relativerisk against all four serotypes” is defined as the relative risk inrelation to the risk of dengue disease irrespective of the serotypebeing responsible for the virologically-confirmed dengue disease and thesubject baseline serostatus.

As used herein, “vaccinating” or “inoculating” refers to theadministration of a vaccine to a subject, with the aim to prevent thesubject, from developing one or more symptoms of a disease. As usedherein, “vaccinating against dengue disease” or “inoculating againstdengue disease” refers to the administration of a dengue vaccinecomposition to a subject, with the aim to prevent the subject, fromdeveloping one or more symptoms of dengue disease. In principle themethod comprises a primary vaccination and optionally one or morebooster vaccinations. The primary vaccination is defined as the primaryadministration schedule for administering the composition or unit doseas disclosed herein to establish a protective immune response and e.g.consists of two administrations e.g. within three months. Whenever anadministration is mentioned within this disclosure such administrationrefers to the primary vaccination unless it is specified as boostervaccination. The booster vaccination refers to an administration oradministration schedule which takes place after the primary vaccinatione.g. at least 1 year or even 5 or 10 years after the lastadministration, e.g. the second administration, of the primaryvaccination schedule. The booster administration attempts at enhancingor reestablishing the immune response of the primary vaccination.

As used herein, the terms “subject” or “subjects” are limited to humansubjects (e.g. infants, children or adults). The terms “elderly subject”or “elderly subjects” refer to subjects with an age of more than 60years, such as 61 years to 100 years, 61 years to 90 years, 61 years to80 years, 61 years to 75 years, or 61 years to 70 years.

As used herein, “subject population” refers to a group of subjects. Thesubject population may refer to least 40 subjects, at least 50 subjects,at least 60 subjects, at least 100 subjects or at least 1000 subjectsand is defined by certain parameters. The parameters that may be used todefine a subject population include, but are not limited to, the age ofthe subjects, whether the subjects are from a dengue endemic region orfrom a dengue non-endemic region and the serostatus of the subjects.

As used herein, “endemic region” refers to a region where a disease orinfectious agent is constantly present and/or usually prevalent in apopulation within this region. As used herein, “non-endemic region”refers to a region from which the disease is absent or in which it isusually not prevalent. Accordingly, a “dengue endemic region” refers togeographic areas in which an infection with dengue virus is constantlymaintained at a baseline level. A “dengue non-endemic region” is ageographic area in which an infection with dengue virus is notconstantly maintained at a baseline level. Accordingly, subjectpopulations or subjects “from a dengue endemic region” or “from a denguenon-endemic region” refer to subject populations or subjects living ingeographic areas as defined above. Whether a geographic area or asubject population is dengue-endemic or not can be determined bydifferent calculatory methods such as the ones described in Bhatt et al.(2013) Nature 496 (7446): 504-507 and supplementary material and inStanaway et al. (2016) Lancet Infect Dis. 16(6): 712-723 andsupplementary material. Overviews of dengue endemic regions and dengueepidemiology are regularly published, for example, by the WHO or CDC.Typical dengue-endemic regions are in Latin America, Southeast Asia andthe Pacific islands and dengue endemic countries include, but are notlimited to, Australia, Brazil, Bangladesh, Colombia, China, DominicanRepublic, Indonesia, India, Mexico, Malaysia, Nicaragua, Nigeria,Pakistan, Panama, Philippines, Puerto Rico, Singapore, Sri Lanka,Thailand and Vietnam. The area's force of infection is measured byseroprevalence surveys provided as seroprevalence rate. Areas with veryhigh force of infection are considered to have a seroprevalence rate ofmore than 80%. As used herein the term “region” when it concernsseroprevalence rates refers to a geographic area where theseroprevalence rate could be determined or is known, e.g. a village, atown, a city, a region, a county, a state, a province or parts of theforegoing or a whole country.

As used herein, “serostatus” refers to the amount of antibodies asubject has with respect to a certain infectious agent, in particulardengue virus. As used herein, “seronegative” or “seronaïve” means thatthe subject does not have neutralizing antibodies against any one ofdengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4 in the serum. Aseronegative or seronaïve subject or subject population is defined by aneutralizing antibody titer of less than 10 for each one of the fourdengue serotypes. A subject or subject population having a neutralizingantibody titer of equal to or more than 10 for at least one dengueserotype is defined as being “seropositive” with respect to said dengueserotype. Serostatus at baseline refers to the serostatus before theadministration of a dengue vaccine composition as described herein.

As used herein, a “neutralizing antibody titer” refers to the amount ofantibodies in the serum of a subject that neutralize the respectivedengue serotype. The neutralizing antibody titer against DENV-1, DENV-2,DENV-3 and DENV-4 is determined in a serum sample of the subject usingknown methods such as the plaque reduction neutralization test (PRNT) asdescribed in the WHO Guidelines (World Health Organization Department ofImmunization Vaccines Biologicals (2007) Guidelines for plaque reductionneutralization testing of human antibodies to dengue viruses,WHO/IVB/07.07) or a microneutralization (MNT50) assay as describedherein. As used herein, the “ratio of not more than 20 for theneutralizing antibody titer of dengue serotype 2 to the neutralizingantibody titer of dengue serotype 4” means that the neutralizingantibody titer of dengue serotype 2 is divided by the neutralizingantibody titer of dengue serotype 4 and that the ratio obtained herebyis no more than 20. In other words, the neutralizing antibody titer ofdengue serotype 2 is not more than 20-times higher than the neutralizingantibody titer of dengue serotype 4 in the subject.

As used herein, the terms “geometric mean neutralizing antibody titer”and “GMT” refer to the geometric mean value of the titer of neutralizingantibodies against the corresponding dengue serotype in the serum ofsubjects in a subject population. The geometric mean value is calculatedby a well-known formula. As used herein, the “ratio of not more than 20for the GMT of dengue serotype 2 to the GMT of dengue serotype 4” meansthat the geometric mean neutralizing antibody titer of dengue serotype 2(GMT DENV-2) is divided by the geometric mean neutralizing antibodytiter of dengue serotype 4 (GMT DENV-4) and that the ratio obtainedhereby is no more than 20. In other words, the geometric meanneutralizing antibody titer of dengue serotype 2 is not more than20-times higher than the geometric mean neutralizing antibody titer ofdengue serotype 4 in the subject population.

As used herein, an “immune response” refers to a subjects response tothe administration of the dengue vaccine. In particular, the immuneresponse includes the formation of neutralizing antibodies to one ormore dengue serotypes. It may also include the stimulation of acell-mediated response or the formation of antibodies to non-structuralproteins such as NS1. An immune response is stimulated by theadministration of a unit dose of the invention as described herein, ifthe titer of neutralizing antibodies against at least one dengue virusserotype and preferably against all four dengue virus serotypes isincreased after said administration of said unit dose. An immuneresponse is stimulated by the administration of a unit dose of theinvention as described herein, if the secretion of interferon gamma byperipheral blood mononuclear cells stimulated with peptides from denguevirus proteins is increased after said administration of said unit dose.An immune response is stimulated by the administration of a unit dose ofthe invention as described herein, if the titer of antibodies tonon-structural proteins such as NS1 is increased after saidadministration of said unit dose. In a particular embodiment, theadministration of a reconstituted unit dose of the present invention asdescribed herein stimulates the formation of neutralizing antibodies toone or more dengue serotypes, a cell-mediated response and the formationof antibodies to non-structural proteins such as NS1.

As used herein, a “balanced immune response” means that the immuneresponse to the four dengue serotypes is sufficient to provideprotection against infection by all four dengue serotypes and preferablythe immune response to the four dengue serotypes has a similar strength.In particular, the neutralizing antibody titer against the four dengueserotypes at day 180 or day 365 after administration of a firstreconstituted unit dose of the invention as described herein is similar,i.e. it differs by less than factor 30, by less than factor 25 or byless than factor 20.

The “total concentration in pfu/0.5 ml” which serves as a base value forthe calculation of the percentage concentration for each individualcomponent of a tetravalent dengue vaccine is shown for one exemplarytetravalent vaccine composition comprising dengue serotype 1 in aconcentration of 3.60 log 10 pfu/0.5 ml, a dengue serotype 2concentration of 4.00 log 10 pfu/0.5 ml, a dengue serotype 3concentration of 4.60 log 10 pfu/0.5 ml and a dengue serotype 4concentration of 5.11 log 10 pfu/0.5 ml.

Primarily, the logarithmic values of the concentrations are convertedinto numerical values. The results of this conversion are 4×10³ pfu/0.5ml for serotype 1, 1×10⁴ pfu/0.5 mll for serotype 2, 4×10⁴ pfu/0.5 mlfor serotype 3 and 1.3×10⁵ pfu/0.5 ml for serotype 4. The totalconcentration in pfu/0.5 ml is the sum of the preceding numerical valuesresulting in 1.84×10⁵ pfu/0.5 ml.

The “percentage concentration” for each of the serotypes 1, 2, 3 and 4is obtained by dividing the numerical concentration value (expressed aspfu/0.5 ml) of an individual serotype by the total concentration(expressed in pfu/0.5 ml) and multiplying the result by 100 i.e.:

Percentage concentration of serotype 1=(4×10³ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=2%

Percentage concentration of serotype 2=(1×10⁴ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=5%

Percentage concentration of serotype 3=(4×10⁴ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=22%

Percentage concentration of serotype 4=(1.3×10⁵ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=71%.

The percentage concentrations are rounded to whole numbers.

As used herein the “concomitant” administration of vaccines refers to acombined administration of two or more vaccines. In the context of partsof the invention, the combined administration of two or more vaccinesrefers to combining the administration schedule of a dengue vaccine,such as the unit dose of the invention, with the administration scheduleof a fellow fever vaccine, such as YF-17D, and/or of a hepatitis Avaccine, such as HAVRIX® or VAQTA®, and/or of a HPV vaccine, such as a9vHPV vaccine, and/or of a combined measles, mumps and rubella (MMR)vaccine, and/or of a combined Tdap vaccine, such as a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, and/or of a DTaP/IPV/HIB vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®.

As used herein “simultaneous” administration means an administration ofat least two different vaccines, such as a dengue vaccine and a fellowfever vaccine, or a dengue vaccine and a hepatitis A vaccine, or adengue vaccine and a HPV vaccine, or a dengue vaccine and a MMR vaccine,or a dengue vaccine and a Tdap vaccine, or a dengue vaccine and aDTaP/IPV/Hib vaccine on the same day. The simultaneous administrationmay be administered by the same medical practitioner, such as during thesame medical appointment.

As used herein “sequential” administration means an administration of atleast two different vaccines, such as a dengue vaccine and a fellowfever vaccine, or a dengue vaccine and a hepatitis A vaccine, or adengue vaccine and a HPV vaccine, or a dengue vaccine and a MMR vaccine,or a dengue vaccine and a Tdap vaccine, or a dengue vaccine and aDTaP/IPV/Hib vaccine on subsequent days, such as within 90 days, but ina combined administration schedule including the administration of thedengue vaccine and the fellow fever vaccine.

As used herein “HPV-associated cancers or genital warts” refers tocancers and genital wards caused by an HPV infection, respectively. TheHPV serotypes 16, 18, 45, 31, 33, 52, 58, also referred to as“high-risk” HPV serotypes, are the types most common in cervicalcancers, wherein the two HPV serotypes 16 and 18 cause about 70% ofcervical cancers worldwide. The HPV serotypes 6 and 11, also referred toas “low-risk” HPV serotypes, cause genital warts.

As used herein a “9vHPV vaccine” refers to a 9-valent HPV vaccine thatprovides protection against the HPV serotypes 6, 11, 16, 18, 31, 33, 45,52, and 58. In particular, the 9vHPV vaccine is used to preventcervical, vulvar, vaginal, and anal cancers, precancerous or dysplasticlesions, and genital warts caused by one or more of these HPV serotypes.

As used herein, a “MMR vaccine” refers to a combined vaccine formeasles, mumps and rubella. Several MMR vaccine are known in the priorart and include M-M-R® II, Priorix®, Tresivac®, and Trimovax®.

As used herein, a “Tdap vaccine” refers to a combined vaccine fortetanus, diphtheria and pertussis. Tdap vaccines known in the prior artinclude INFANRIX® (for vaccination of children from 6 weeks to 7 yearsof age), and BOOSTRIX® from GlaxoSmithKline and Adacel from SanofiPasteur (both for use in individuals of 10 years of age or older).

As used herein, “DTaP” refers to diphtheria, tetanus and acellularpertussis.

As used herein, “IPV” refers to inactivated poliovirus.

As used herein, “Hib” refers to Haemophilus influenzae type b.

As used herein, a “DTaP/IPV/Hib vaccine” refers to a combined vaccinefor diphtheria, tetanus, pertussis, poliomyelitis and Haemophilusinfluenzae type b. A DTaP/IPV/Hib vaccine known in the prior artincludes Pentacel® from Sanofi Pasteur.

As used herein, the term “chronic disease or condition” includes thosediseases and conditions which persist in an elderly subject for threemonths or more. In particular, it includes diabetes, hypertension,allergies, previous strokes, ischemic heart disease, chronic renalimpairment and chronic obstructive pulmonary disease.

As used herein, the term “impaired immune system” means that at leastone function of at least one component of the immune system is weakerthan in younger subjects, i.e. in subjects with an age of less than 60years. These functions include a lower antioxidant response of monocytesagainst oxidative stress induced by dengue virus and lower T cellresponses and cytokine production in response to dengue virus infection.

As used herein, “solicited systemic adverse events” in children under 6years are defined as fever, irritability/fussiness, drowsiness and lossof appetite that occurred within 14 days after each vaccination, and inchildren of 6 years or more are defined as fever, headache, asthenia,malaise and myalgia that occurred within 14 days after each vaccination.

As used herein, “solicited local adverse events” are injection sitepain, injection site erythema and injection site swelling that occurredwithin 7 days after each vaccination.

As used herein, “unsolicited adverse events” are any adverse events(AEs) that are not solicited local or systemic AEs, as defined above.

As used herein, a “serious adverse event” or “SAE” is any untowardmedical occurrence or effect that at any dose results in death, islife-threatening, requires inpatient hospitalization or prolongation ofexisting hospitalization, results in persistent or significantdisability/incapacity, is a congenital anomaly/birth defect or ismedically important due to other reasons than the above mentionedcriteria.

The relationship of each AE, including solicited systemic AEs (solicitedlocal AEs are considered as related) to trial vaccine(s) will beassessed using the following categories: As used herein, “IP-Related AE”or “vaccine related AE” means that there is suspicion that there is arelationship between the vaccine and the AE (without determining theextent of probability); there is a reasonable possibility that thevaccine contributed to the AE. As used herein, “Non-IP Related” or“non-vaccine related” means that there is no suspicion that there is arelationship between the vaccine and the AE; there are other more likelycauses and administration of the vaccine is not suspected to havecontributed to the AE.

As used herein, a subject or subject population being “2 to 17 years ofage” refers to a subject or subject population being 2 to 17 years ofage on the first day of the administration of the dengue vaccinecomposition as described herein.

As used herein “%-points” refers to the difference of two %-values in a%-value. For example two values in % which are within 5%-points refersto e.g. one value at 1% and a second value at 6%.

As used herein, the term “determination of the previous dengue infectionin the subject before administration” means that a previous dengueinfection has to be assessed before vaccination in that there is alaboratory confirmed history of dengue or through an appropriatelyvalidated serological test e.g. by the method as disclosed herein suchas the MNT50 test described in Example 2 or any serotesting withadequate performance in terms of specificity and cross reactivity basedon the locale disease epidemiology.

As used herein % w/v refers to % mg/ml wherein e.g. 150 mg/ml are 15%w/v.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Genetic structure of the four dengue strains contained in TDV.The solid red triangles indicate the three attenuating mutations presentin the 5′NCR, NS1 and NS3 proteins. The TDV-1, TDV-3 and TDV-4 strainsare chimeric viruses where the prM and E genes from dengue serotype 1, 3and 4, respectively, are inserted into the TDV-2 backbone.

FIG. 2: Schematic drawing illustrating the microneutralization test(MNT) used to determine the titer of neutralizing antibodies.

FIG. 3: Percentage of subjects (±95% confidence intervals) who wereseropositive (reciprocal neutralizing titer 10) for each of the dengueserotypes at different time points of the trial in the HD-TDV group(dark colored, left bar) and TDV group (light colored, right bar)throughout the trial. Time points shown are baseline, day 15 (d15), day30 (d30), day 90 (d90), day 180 (d180) and day 365 (d365). Part A showsthe results for participants seropositive (set of graphs on the left)and seronegative (set of graphs on the right) at baseline, per-protocolset. Part B shows the results for the entire trial population (all)per-protocol set

FIG. 4: Geometric mean titers (GMTs) (±95% confidence intervals) ofneutralizing antibodies against each of the four dengue serotypes duringthe course of the trial for HD-TDV (dark line with triangles) and TDV(light line with circles) recipients, for the entire trial population(part B) and for participants seropositive and seronegative at baseline(part A), per-protocol set.

FIG. 5: IFNγ ELISpot analysis of peripheral blood mononuclear cellsbefore vaccination (baseline) and at different time points afteradministration of TDV. Shown are the response frequencies to the entireDENV-2 proteome. A subject was considered responsive if response to morethan one peptide pool from DENV-2 was positive (i.e. ≥4×mean of negativecontrol and ≥50 SFC/10⁶ PBMCs).

FIG. 6: IFNγ ELISpot analysis of peripheral blood mononuclear cellsbefore vaccination (baseline) and at different time points afteradministration of TDV. Shown are the response frequencies to peptidepools matching selected DENV-derived proteins as indicated. A subjectwas considered responsive if response to more than one peptide pool fromDENV-2 was positive (i.e. ≥4×mean of negative control and ≥50 SFC/10⁶PBMCs). A=DENV-2 C; B=DENV-1 prM+E; C=DENV-2 prM+E; D=DENV-3 prM+E;E=DENV-4 prM+E; F=DENV-2 NS1; G=DENV-2 NS2; H=DENV-2 NS3; I=DENV-2 NS4;J=DENV-2 NS5.

FIG. 7: Effect of sera from a seronegative subject (A) and aseropositive subject (A) to whom TDV was administered on DENV-2NS1-induced hyperpermeability as determined by TEER. HPMEC were grown onTranswell semi-permeable membranes (0.4 μm pore size), and serum samples(30 μl) were added to the apical chamber in the presence or absence ofDENV2 NS1 (5 μg/ml). DENV2 NS1 is depicted as squares; day 0 serum aloneis depicted as diamonds; day 120 serum alone is depicted as triangles;day 0 serum+DENV2 NS1, is depicted as upside-down triangles; day 120serum+DENV2 NS1 is depicted as X's. ({circumflex over ( )}) representsmedia change. Endothelial permeability was measured at indicatedtime-points over 48 hours. Relative TEER values from one independentexperiment performed in duplicate are plotted. Error bars indicatestandard error of the mean (SEM).

FIG. 8: Effect of sera from seronegative and seropositive subjects towhich TDV was administered on NS1-induced sialic acid and heparansulfate degradation. Shown is the quantification of mean fluorescenceintensity (MFI) of (A) sialic acid and (B) heparan sulfate expressionafter staining with sialic acid- and heparan sulfate-specificfluorescent antibodies as visualized by confocal microscopy. Values arenormalized to MFI from the NS1+positive control serum group (representedby dotted line at 100%) and expressed as percentage of control. Errorbars indicate SEM. The left bar for each subject shows the results atday 0 (d0), the right car for each subject shows the results at day 120(d120).

FIG. 9: Flow diagram of the clinical trial of Example 6.

FIG. 10: Cumulative incidence of A) virologically-confirmed dengue casesand B) hospitalized virologically-confirmed dengue cases over timeduring Part 1 study period by baseline serostatus (safety set data; datapresented truncated at Month 18). Tables show numbers of participantsunder follow-up at various time points to end of Part 1 study period.

FIG. 11: Study design of phase III study described in example 6

DETAILED DESCRIPTION Dengue Virus Strains

The dengue virus is a single stranded, positive sense RNA virus of thefamily flaviviridae. The taxonomy is outlined in Table 1. The familyflaviviridae includes three genera, flavivirus, hepacivirus andpestivirus. The genus flavivirus contains highly pathogenic andpotentially hemorrhagic fever viruses, such as yellow fever virus anddengue virus, encephalitic viruses, such as Japanese encephalitis virus,Murray Valley encephalitis virus and West Nile virus, and a number ofless pathogenic viruses.

TABLE 1 Dengue Virus Taxonomy of the GMO Parental Strain FamilyFlaviviridae Genus Flavivirus Species Dengue virus Strains DengueSerotype 2 (Strain 16681), Strain DEN-2 PDK-53 GMO parent TDV-2

The flavivirus genome comprises in 5′ to 3′ direction (see FIG. 1):

a 5′-noncoding region (5′-NCR),

a capsid protein (C) encoding region,

a pre-membrane protein (prM) encoding region,

an envelope protein (E) encoding region,

a region encoding nonstructural proteins (NSI, NS2A, NS2B, NS3, NS4A,NS4B, NS5) and

a 3′ noncoding region (3′-NCR).

The viral structural proteins are C, prM and E, and the nonstructuralproteins are NSI to NS5. The structural and nonstructural proteins aretranslated as a single polyprotein and processed by cellular and viralproteases.

The unit dose of the invention as described herein comprises a denguevirus composition that comprises four live attenuated dengue virusstrains (tetravalent dengue virus composition) representing dengueserotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4.Preferably the composition comprises chimeric dengue viruses andoptionally at least one non-chimeric dengue virus, in particular amolecularly characterized and cloned dengue serotype 2 strain derivedfrom the live attenuated DEN-2 PDK-53 virus strain (TDV-2), and threechimeric dengue strains derived from the TDV-2 strain by replacing thestructural proteins prM and E from TDV-2 with the correspondingstructural proteins from the other dengue serotypes, resulting in thefollowing chimeric dengue strains:

a DENV-2/1 chimera (TDV-1),

a DENV-2/3 chimera (TDV-3) and

a DENV-2/4 chimera (TDV-4).

The genetically modified tetravalent dengue vaccine TDV is based on amolecularly characterized and cloned dengue-2 virus strain (TDV-2). Thisattenuated TDV-2 strain was generated by cDNA cloning of the attenuatedlaboratory-derived DEN-2 PDK-53 virus strain that was originallyisolated at Mahidol University, Bangkok, Thailand (Kinney et al. (1997)Virology 230(2): 300-308). DEN-2 PDK-53 was generated by 53 serialpassages in primary dog kidney (PDK) cells at 32° C. (Bhamarapravati etal. (1987) Bull. World Health Organ. 65(2): 189-195).

The attenuated DEN-2 PDK-53 strain (the precursor of TDV-2) was derivedfrom the wild type virus strain DEN-2 16681 (SEQ ID NO 11) and differsin nine nucleotides from the wild type as follows (Kinney et al. (1997)Virology 230(2): 300-308):

-   -   (i) 5′-noncoding region (NCR)-57 (nt-57 C-to-T): major        attenuation locus    -   (ii) prM-29 Asp-to-Val (nt-524 A-to-T)    -   (iii) nt-2055 C-to-T (E gene) silent mutation    -   (iv) NS1-53 Gly-to-Asp (nt-2579 G-to-A): major attenuation locus    -   (v) NS2A-181 Leu-to-Phe (nt-4018 C-to-T)    -   (vi) NS3-250 Glu-to-Val (nt-5270 A-to-T): major attenuation        locus    -   (vii) nt-5547 (NS3 gene) T-to-C silent mutation    -   (viii) NS4A-75 Gly-to-Ala (nt-6599 G-to-C)    -   * nt-8571 C-to-T (NS5 gene) silent mutation

The three nucleotide changes located in the 5′ noncoding region (NCR)(nucleotide 57) (mutation (i)), the NS-1 (amino acid 828 of SEQ ID NO.4) (mutation (iv)) and NS-3 genes (amino acid 1725 of SEQ ID NO. 4)(mutation (vi)) form the basis for the attenuation phenotype of theDEN-2 PDK-53 strain (Butrapet et al. (2000) J. Virol. 74(7): 3111-3119)(Table 2). These three mutations are referred to herein as the“attenuating mutations” and are comprised in TDV-1, TDV-2, TDV-3 andTDV-4.

TABLE 2 Attenuating mutations in the common genetic backbone of all TDVstrains Location of Mutation Nucleotide Change in TDV-2 Amino AcidChange in TDV-2 5′ Noncoding Region (5′NCR) 57 C to T Not applicable(silent) Nonstructural Protein 1 (NS1) 2579 G to A 828 Gly to AspNonstructural Protein 3 (NS3) 5270 A to T 1725 Glu to Val

In one embodiment, TDV-2 comprises in addition to the three attenuatingmutations one or more mutations selected from:

a) a mutation in the prM gene at nucleotide 524 from adenine tothymidine resulting in an amino acid change at position 143 fromasparagine to valine, and/or

b) a silent mutation in the E gene at nucleotide 2055 from cytosine tothymidine, and/or

c) a mutation in the NS2A gene at nucleotide 4018 from cytosine tothymidine resulting in an amino acid change at position 1308 fromleucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5547 from thymidineto cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6599 from guanine tocytosine resulting in an amino acid change at position 2168 from glycineto alanine, and/or

f) a silent mutation in the prM gene at nucleotide 900 from thymidine tocytosine.

The silent mutation in the NS5 gene at nucleotide 8571 from cytosine tothymidine of DEN-2 PDK-53 is not present in the TDV-2 strain.

In another embodiment, TDV-2 comprises in addition to the threeattenuating mutations one or more mutations selected from:

g) a mutation in the prM gene at nucleotide 592 from adenine to guanineresulting in an amino acid change at position 166 from lysine toglutamine, and/or

h) a mutation in the NS5 gene at nucleotide 8803 from adenine to guanineresulting in an amino acid change at position 2903 from isoleucine tovaline.

In another embodiment, TDV-2 comprises in addition to the threeattenuating mutations the mutations a) and g), preferably the mutationsa), g), c), e) and h), more preferably the mutations a), g), c), e), h)and b), even more preferably the mutations a), g), c), e), h), b) andd), and most preferably the mutations a) to h). The nucleotide positionsand amino acids positions of TDV-2 refer to the nucleotide sequence asshown in SEQ ID NO. 3 and amino acid sequence as shown in SEQ ID NO. 4.

The dengue virus structural envelope (E) protein and pre-membrane (prM)protein have been identified as the primary antigens that elicit aneutralizing protective antibody response (Plotkin 2001). For creationof the tetravalent dengue vaccine (TDV), TDV-2 was modified by replacingthe nucleic acid sequence encoding the DENV-2 prM and E glycoproteinswith the nucleic acid sequence encoding the corresponding wild type prMand E glycoproteins from the DENV-1, DENV-3, and DENV-4 wild typestrains DENV-1 16007, DENV-3 16562 or DENV-4 1036 virus, respectively,(see Table 3) using standard molecular genetic engineering methods(Huang et al. (2003) J. Virol. 77(21): 11436-11447).

TABLE 3 Viral origin of prM/E gene regions of the TDV virus strainsNucleotide Amino acid Virus Strain Origin Source Reference sequencesequence DENV-1 16007 Thailand, 1964 DHF/DSS Halstead and SEQ ID NO. 9SEQ ID NO. 10 patient Simasthien, 1970 DENV-2 16681 Thailand, 1964DHF/DSS Halstead and SEQ ID NO. 11 SEQ ID NO. 12 patient Simasthien,1970 DENV-3 16562 Philippines, 196 DHF patient Halstead and SEQ ID NO.13 SEQ ID NO. 14 Simasthien, 1970 DENV-4 1036 Indonesia, 1976 DF patientGubler et al., 1979 SEQ ID NO. 15 SEQ ID NO. 16

A diagram of the four TDV strains comprised in the dengue vaccinecomposition is shown in FIG. 1.

The chimeric dengue strains TDV-1, TDV-3 and TDV-4 express the surfaceantigens prM and E of the DENV-1, DENV-3 or DENV-4 viruses, as depictedin Table 3 respectively, and retain the genetic alterations responsiblefor the attenuation of TDV-2. Thus, each of the TDV-1, TDV-3 and TDV-4strains comprises the attenuating mutations described in Table 2.

In one embodiment, TDV-1 comprises in addition to the three attenuatingmutations one or more mutations selected from:

c) a mutation in the NS2A gene at nucleotide 4018 from cytosine tothymidine resulting in an amino acid change at position 1308 fromleucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5547 from thymidineto cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6599 from guanine tocytosine resulting in an amino acid change at position 2168 from glycineto alanine, and/or

i) a silent mutation in the E gene at nucleotide 1575 from thymidine tocytosine, and/or

j) a silent mutation in the junction site between the prM-E gene and theDEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and/or

k) a mutation in the junction site between the prM-E gene and the DEN-2PDK-53 backbone at nucleotides 2381/2382 from thymidine-guanine tocytosine-cytosine resulting in an amino acid change at position 762 fromvaline to alanine.

In another embodiment, TDV-1 comprises in addition to the threeattenuating mutations one or more mutations selected from:

l) a mutation in the NS2A gene at nucleotide 3823 from adenine tocytosine resulting in an amino acid change at position 1243 fromisoleucine to leucine, and/or

m) a mutation in the NS2B gene at nucleotide 4407 from adenine tothymidine resulting in an amino acid change at position 1437 fromglutamine to asparagine, and/or

n) a silent mutation in the NS4B gene at nucleotide 7311 from adenine toguanine.

In another embodiment, the TDV-1 strain comprises in addition to thethree attenuating mutations the mutations l) and m), preferably themutations l), m), c) and e), even more preferably the mutations l), m),c), e), d) and n), and most preferably the mutations l), m), c), e), d),n), i), j) and k). The nucleotide positions and amino acids positions ofTDV-1 refer to the nucleotide sequence as shown in SEQ ID NO. 1 andamino acid sequence as shown in SEQ ID NO. 2.

In one embodiment, TDV-3 comprises in addition to the three attenuatingmutations one or more mutations selected from:

c) a mutation in the NS2A gene at nucleotide 4012 from cytosine tothymidine resulting in an amino acid change at position 1306 fromleucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5541 from thymidineto cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6593 from guanine tocytosine resulting in an amino acid change at position 2166 from glycineto alanine, and/or

j) a silent mutation in the junction site between the prM-E gene and theDEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and/or

k) a mutation in the junction site between the prM-E gene and the DEN-2PDK-53 backbone at nucleotides 2375/2376 from thymidine-guanine tocytosine-cytosine resulting in an amino acid change at position 760 fromvaline to alanine, and/or

o) a silent mutation in the prM gene at nucleotide 552 from cytosine tothymidine, and/or

p) a mutation in the E gene at nucleotide 1970 from adenine to thymidineresulting in an amino acid change at position 625 from histidine toleucine.

In another embodiment, TDV-3 comprises in addition to the threeattenuating mutations one or more mutations selected from:

q) a mutation in the E gene at nucleotide 1603 from adenine to thymidineresulting in an amino acid change at position 503 from threonine toserine, and/or

r) a silent mutation in the NS5 gene at nucleotide 7620 from adenine toguanine.

In another embodiment, TDV-3 comprises in addition to the threeattenuating mutations the mutations p) and q), preferably the mutationsp), q), c) and e), even more preferably the mutations p), q), c), e), d)and r), and most preferably the mutations p), q), c), e), d), r), j), k)and o). The nucleotide positions and amino acids positions of TDV-3refer to the nucleotide sequence as shown in SEQ ID NO. 5 and amino acidsequence as shown in SEQ ID NO. 6.

In one embodiment, TDV-4 comprises in addition to the three attenuatingmutations one or more mutations selected from:

c) a mutation in the NS2A gene at nucleotide 4018 from cytosine tothymidine resulting in an amino acid change at position 1308 fromleucine to phenylalanine, and/or

d) a silent mutation in the NS3 gene at nucleotide 5547 from thymidineto cytosine, and/or

e) a mutation in the NS4A gene at nucleotide 6599 from guanine tocytosine resulting in an amino acid change at position 2168 from glycineto alanine, and/or

j) a silent mutation in the junction site between the prM-E gene and theDEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and/or

k) a mutation in the junction site between the prM-E gene and the DEN-2PDK-53 backbone at nucleotides 2381/2382 from thymidine-guanine tocytosine-cytosine resulting in an amino acid change at position 762 fromvaline to alanine, and/or

s) a mutation in the C gene at nucleotide 396 from adenine to cytosineresulting in an amino acid change at position 100 from arginine toserine, and/or

t) a silent mutation in the E gene at nucleotide 1401 from adenine toguanine, and/or

u) a mutation in the E gene at nucleotide 2027 from cytosine tothymidine resulting in an amino acid change at position 644 from alanineto valine, and/or

v) a mutation in the E gene at nucleotide 2275 from adenine to cytosineresulting in an amino acid change at position 727 from methionine toleucine.

In another embodiment, TDV-4 comprises in addition to the threeattenuating mutations one or more mutations selected from:

w) a silent mutation in the C gene at nucleotide 225 from adenine tothymidine, and/or

x) a mutation in the NS2A gene at nucleotide 3674 from adenine toguanine resulting in an amino acid change at position 1193 fromasparagine to glycine, and/or

y) a mutation in the NS2A gene at nucleotide 3773 from adenine to anadenine/guanine mix resulting in an amino acid change at position 1226from lysine to a lysine/asparagine mix, and/or

z) a silent mutation in the NS3 gene at nucleotide 5391 from cytosine tothymidine, and/or

aa) a mutation in the NS4A gene at nucleotide 6437 from cytosine tothymidine resulting in an amino acid change at position 2114 fromalanine to valine, and/or

bb) a silent mutation in the NS4B gene at nucleotide 7026 from thymidineto a thymidine/cytosine mix, and/or

cc) a silent mutation in the NS5 gene at nucleotide 9750 from adenine tocytosine.

In another embodiments, TDV-4 comprises in addition to the threeattenuating mutations the mutation s), u) and v), preferably themutations s), u), v), c), e), x), y) and aa), even more preferably themutations s), u), v), c), e), x), y), aa) and w), even more preferablythe mutations s), u), v), c), e), x), y), aa), w), d), z), bb) and cc),and most preferably the mutations s), u), v), c), e), x), y), aa), w),d), z), bb), cc), j), k) and t). The nucleotide positions and aminoacids positions of TDV-4 refer to the nucleotide sequence as shown inSEQ ID NO. 7 and amino acid sequence as shown in SEQ ID NO. 8.

In a preferred embodiment, TDV-1 has the nucleotide sequence of SEQ IDNO. 1, TDV-2 has the nucleotide sequence of SEQ ID NO. 3, TDV-3 has thenucleotide sequence of SEQ ID NO. 5, and/or TDV-4 has the nucleotidesequence of SEQ ID NO. 7. In a further preferred embodiment, TDV-1 hasthe amino acid sequence of SEQ ID NO. 2, TDV-2 has the amino acidsequence of SEQ ID NO. 4, TDV-3 has the amino acid sequence of SEQ IDNO. 6, and TDV-4 has the amino acid sequence of SEQ ID NO. 8. In afurther preferred embodiment, TDV-1 has a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO. 2, TDV-2 has a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO. 4, TDV-3 has a nucleotidesequence encoding the amino acid sequence of SEQ ID NO. 6, and TDV-4 hasa nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 8.

TABLE 4 Sequences of the TDV virus strains SEQ ID NO. dengue virusstrain sequence type SEQ ID NO. 1 TDV-1 nucleotide sequence SEQ ID NO. 2TDV-1 amino acid sequence SEQ ID NO. 3 TDV-2 nucleotide sequence SEQ IDNO. 4 TDV-2 amino acid sequence SEQ ID NO. 5 TDV-3 nucleotide sequenceSEQ ID NO. 6 TDV-3 amino acid sequence SEQ ID NO. 7 TDV-4 nucleotidesequence SEQ ID NO. 8 TDV-4 amino acid sequence

Thus, in a particularly preferred embodiment, the unit dose of theinvention as described herein comprises the live attenuated dengue virusstrains TDV-1, TDV-2, TDV-3 and TDV-4, wherein TDV-1, TDV-3 and TDV-4are based on TDV-2 and comprise the prM and E regions of DENV-1, -3 and-4, respectively. In another particularly preferred embodiment, TDV-1 ischaracterized by the nucleotide sequence according to SEQ ID No. 1 andthe amino acid sequence according to SEQ ID No. 2, TDV-2 ischaracterized by the nucleotide sequence according to SEQ ID No. 3 andthe amino acid sequence according to SEQ ID No. 4, TDV-3 ischaracterized by the nucleotide sequence according to SEQ ID No. 5 andthe amino acid sequence according to SEQ ID No. 6 and TDV-4 ischaracterized by the nucleotide sequence according to SEQ ID No. 7 andthe amino acid sequence according to SEQ ID No. 8.

The E protein of DENV-3 has two fewer amino acids than the E protein ofDENV-2. Therefore, the nucleotides and encoded amino acid backbone ofTDV-2 starting after the E region of DENV-3 at nucleotide 2374 of SEQ IDNO. 5 and amino acid 760 of SEQ ID NO. 6 are 6 nucleotides less and 2amino acids less than the original TDV-2 nucleotide and amino acidpositions, respectively.

Dengue Vaccine Composition

The present invention is in part directed to a unit dose of a denguevaccine composition as described. The dengue vaccine compositioncomprises a tetravalent dengue virus composition, also referred to asdengue virus composition, and pharmaceutically acceptable excipients.

Dengue Virus Composition, Virus Concentrations and %-Concentrations

The present invention is in part directed to a unit dose of a denguevaccine composition, wherein the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains:

-   -   (i) a dengue serotype 1 preferably in a concentration of at        least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 preferably in a concentration of at        least 2.7 log 10 pfu/0.5 mL,    -   (iii) a dengue serotype 3 preferably in a concentration of at        least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a dengue serotype 4 preferably strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.

In one embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains:

-   -   (i) a dengue serotype 1 preferably in a concentration of at        least 3.3 log 10 pfu/0.5 mL to 3.8 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 preferably in a concentration of at        least 2.7 log 10 pfu/0.5 mL,    -   (iii) a dengue serotype 3 preferably in a concentration of at        least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a dengue serotype 4 preferably strain in a concentration of        at least 4.5 log 10 pfu/0.5 ml or 4.6 log 10 pfu/0.5 mL,        optionally to 6.2 log 10 pfu/0.5 ml.

The present invention is further in part directed to a unit dose of adengue vaccine composition, wherein the dengue vaccine compositioncomprises a tetravalent dengue virus composition including four liveattenuated dengue virus strains:

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.

In one embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains:

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL to 3.8 log 10 pfu/0.5 ml,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL or at least 4.6 log 10 pfu/0.5 mL        to optionally 6.2 log 10 pfu/0.5 ml.

Preferably, the chimeric dengue serotype 2/1 strain is TDV-1, the dengueserotype 2 strain is TDV-2, the chimeric dengue serotype 2/3 strain isTDV-3 and the chimeric dengue serotype 2/4 strain is TDV-4.

In one embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/0.5 mL to 5.3 log        10 pfu/0.5 mL,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/0.5 mL to 5.0 log 10 pfu/0.5 mL,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/0.5 mL to 6.0 log        10 pfu/0.5 mL, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/0.5 mL to 6.5 log        10 pfu/0.5 mL.

In one such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/0.5 mL to 5.0 log        10 pfu/0.5 mL,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/0.5 mL to 4.9 log 10 pfu/0.5 mL,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/0.5 mL to 5.7 log        10 pfu/0.5 mL, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/0.5 mL to 6.2 log        10 pfu/0.5 mL.

In a further such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 5.0 log 10        pfu/dose,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 4.9 log 10 pfu/dose,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 5.7 log 10        pfu/dose, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 5.5 log 10        pfu/dose.

In a further such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 4.1 log 10        pfu/dose,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 3.6 log 10 pfu/dose,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 4.7 log 10        pfu/dose, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 5.3 log 10        pfu/dose.

In a further such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/0.5 mL to 3.6 log        10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/0.5 mL to 4.0 log 10 pfu/0.5 mL,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/0.5 mL to 4.6 log        10 pfu/0.5 mL, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/0.5 ml or 4.6 log        10 pfu/0.5 mL to 5.1 log 10 pfu/0.5 mL.

In another embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 4.3 log 10 pfu/0.5 mL to 4.4 log        10 pfu/0.5 mL,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 3.7 log 10 pfu/0.5 mL to 3.8 log 10 pfu/0.5 mL,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.5 log 10 pfu/0.5 mL to 5.0 log        10 pfu/0.5 mL, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 5.5 log 10 pfu/0.5 mL to 5.6 log        10 pfu/0.5 mL.

In a particularly preferred embodiment, the dengue vaccine compositioncomprises a tetravalent dengue virus composition including four liveattenuated dengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 4.4 log 10 pfu/0.5 mL,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 3.8 log 10 pfu/0.5 mL,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.5 log 10 pfu/0.5 mL, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 5.6 log 10 pfu/0.5 mL.

In another particularly preferred embodiment, the dengue vaccinecomposition comprises a tetravalent dengue virus composition includingfour live attenuated dengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.6 log 10 pfu/0.5 mL,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 4.0 log 10 pfu/0.5 mL,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.6 log 10 pfu/0.5 mL, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 5.1 log 10 pfu/0.5 mL.

In another preferred embodiment, the dengue vaccine compositioncomprises a tetravalent dengue virus composition including four liveattenuated dengue virus strains wherein the arithmetic sum of all fourserotypes is less than 6.7 log 10 pfu/0.5 mL, preferably less than 5.5log 10 pfu/0.5 mL. In certain such embodiments, the arithmetic sum ofall four serotypes is at least 4.6 log 10 pfu/0.5 mL. In a preferredembodiment, the dengue vaccine composition comprises a tetravalentdengue virus composition including four live attenuated dengue virusstrains wherein the arithmetic sum of all four serotypes is in the rangeof 4.6 log 10 pfu/0.5 mL to 6.7 log 10 pfu/0.5 mL, preferably in therange of 4.6 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL.

Preferably, in said embodiments the chimeric dengue serotype 2/1 strainis TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengueserotype 2/3 strain is TDV-3 and the chimeric dengue serotype 2/4 strainis TDV-4. More preferably, TDV-1 is characterized by the nucleotidesequence according to SEQ ID No. 1 and the amino acid sequence accordingto SEQ ID No. 2, TDV-2 is characterized by the nucleotide sequenceaccording to SEQ ID No. 3 and the amino acid sequence according to SEQID No. 4, TDV-3 is characterized by the nucleotide sequence according toSEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 andTDV-4 is characterized by the nucleotide sequence according to SEQ IDNo. 7 and the amino acid sequence according to SEQ ID No. 8.

The present invention is in part directed to a unit dose of a denguevaccine composition, wherein the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) in a concentration of at least 3.3 log 10 pfu/dose,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) in a        concentration of at least 2.7 log 10 pfu/dose,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) in a concentration of at least 4.0 log 10 pfu/dose, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) in a concentration of at least 4.5 log 10 pfu/dose.

In one embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 5.3 log 10        pfu/dose,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 5.0 log 10 pfu/dose,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 6.0 log 10        pfu/dose, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 6.5 log 10        pfu/dose.

In one such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 5.0 log 10        pfu/dose,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 4.9 log 10 pfu/dose,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 5.7 log 10        pfu/dose, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 6.2 log 10        pfu/dose.

In a further such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 5.0 log 10        pfu/dose,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 4.9 log 10 pfu/dose,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 5.7 log 10        pfu/dose, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 5.5 log 10        pfu/dose.

In a further such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 4.1 log 10        pfu/dose,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 3.6 log 10 pfu/dose,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 4.7 log 10        pfu/dose, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 5.3 log 10        pfu/dose.

In a further such embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 3.6 log 10        pfu/dose,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 4.0 log 10 pfu/dose,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 4.6 log 10        pfu/dose, and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose 4.6 log 10        pfu/dose to 5.1 log 10 pfu/dose.

In another embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 4.3 log 10 pfu/dose to 4.4 log 10        pfu/dose,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 3.7 log 10 pfu/dose to 3.8 log 10 pfu/dose,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.5 log 10 pfu/dose to 5.0 log 10        pfu/dose, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 5.5 log 10 pfu/dose to 5.6 log 10        pfu/dose.

In a particularly preferred embodiment, the dengue vaccine compositioncomprises a tetravalent dengue virus composition including four liveattenuated dengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 4.4 log 10 pfu/dose,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 3.8 log 10 pfu/dose,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.5 log 10 pfu/dose, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 5.6 log 10 pfu/dose.

In another particularly preferred embodiment, the dengue vaccinecomposition comprises a tetravalent dengue virus composition includingfour live attenuated dengue virus strains wherein:

-   -   (i) the dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.6 log 10 pfu/dose,    -   (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 4.0 log 10 pfu/dose,    -   (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.6 log 10 pfu/dose, and    -   (iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 5.1 log 10 pfu/dose.

In another preferred embodiment, the dengue vaccine compositioncomprises a tetravalent dengue virus composition including four liveattenuated dengue virus strains wherein the arithmetic sum of all fourserotypes is less than 6.7 log 10 pfu/dose, preferably less than 5.5 log10 pfu/dose. In certain such embodiments, the arithmetic sum of all fourserotypes is at least 4.6 log 10 pfu/dose. in a preferred embodiment,the dengue vaccine composition comprises a tetravalent dengue viruscomposition including four live attenuated dengue virus strains whereinthe arithmetic sum of all four serotypes is in the range of 4.6 log 10pfu/dose to 6.7 log 10 pfu/dose, preferably in the range of 4.6 log 10pfu/dose to 5.5 log 10 pfu/dose.

In one embodiment in the composition (i), (ii), (iii), and (iv) providea total concentration of pfu/0.5 mL and based on said concentration, theconcentration of (iii) at least 10% of the total concentration inpfu/0.5 mL.

In one embodiment in the composition (i), (ii), (iii), and (iv) providea total concentration of pfu/0.5 mL and based on said totalconcentration the concentration of (ii) in pfu/0.5 mL is less than 10%,and the concentration of (iv) in pfu/0.5 mL is at least 50%, and theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 8%, or at least 10%, or at least 12%,or at least 14%, or at least 16%, or at least 18%.

It is preferred that the concentration in the reconstituted unit dose of(iii) in pfu/0.5 mL is at least 10%.

In one embodiment in the composition (i), (ii), (iii), and (iv) providea total concentration of pfu/0.5 mL and based on said totalconcentration the concentration of (ii) in pfu/0.5 mL is less than 2%,the concentration of (iv) in pfu/0.5 mL is at least 50%, theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 6%.

Preferably, in said embodiments the chimeric dengue serotype 2/1 strainis TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengueserotype 2/3 strain is TDV-3 and the chimeric dengue serotype 2/4 strainis TDV-4. More preferably, TDV-1 is characterized by the nucleotidesequence according to SEQ ID No. 1 and the amino acid sequence accordingto SEQ ID No. 2, TDV-2 is characterized by the nucleotide sequenceaccording to SEQ ID No. 3 and the amino acid sequence according to SEQID No. 4, TDV-3 is characterized by the nucleotide sequence according toSEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 andTDV-4 is characterized by the nucleotide sequence according to SEQ IDNo. 7 and the amino acid sequence according to SEQ ID No. 8.

The concentration of the different dengue viruses is preferablydetermined by an immuno-focus assay known in the art. For example, theconcentration may be determined by an immuno-focus assay wherein serialdilutions of dengue virus are applied to monolayers of adherent cells,such as Vero cells. After a period of time which allows infectiousviruses to bind to the cells and to be taken up by the cells, an overlaycontaining thickening agents, such as agarose or carboxymethylcellulose,is added to prevent diffusion of viruses so that progeny viruses canonly infect cells adjacent to the original infected cells. After aperiod of incubation to allow viral replication, cells are fixed andstained using serotype-specific anti-dengue monoclonal antibodies and asecondary antibody such as an antibody labeled with alkalinephosphatase. The foci are stained by adding a suitable substrate for theenzyme attached to the secondary antibody, such as5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium phosphatasesubstrate. The number of plaques on the plate corresponds to the plaqueforming units of the virus in the solutions applied to the cells. Forexample, a concentration of 1,000 pfu/μl indicates that 1 μl of thesolution applied to the cells contains enough viruses to produce 1,000plaques in a cell monolayer.

The dengue vaccine composition comprises a tetravalent dengue viruscomposition including four live attenuated dengue virus strains, whereina chimeric dengue serotype 2/1 strain, a dengue serotype 2 strain, achimeric dengue serotype 2/3 strain, and a chimeric dengue serotype 2/4strain provide a total concentration in pfu/0.5 mL. The term “totalconcentration in pfu/0.5 mL” or “total concentration in pfu/dose” is thesum of the concentrations of the dengue serotype 1 (e.g. chimeric dengueserotype 2/1 strain), dengue serotype 2 (e.g. the dengue serotype 2strain), the dengue serotype 3 (e.g. chimeric dengue serotype 2/3strain) and the dengue serotype 4 (e.g. chimeric dengue serotype 2/4strain), preferably the sum of the concentrations of TDV-1, TDV-2, TDV-3and TDV-4, and is defined as 100% of the dengue virus concentration asdetermined by pfu (plaque forming units) in 0.5 mL or in a dose.

In one embodiment, the dengue vaccine composition comprises atetravalent dengue virus composition including four live attenuateddengue virus strains, wherein a dengue serotype 1 (e.g. chimeric dengueserotype 2/1 strain), a dengue serotype 2 (e.g. dengue serotype 2strain), a dengue serotype 3 (e.g. chimeric dengue serotype 2/3 strain),and a dengue serotype 4 (e.g. chimeric dengue serotype 2/4 strain)provide a total concentration in pfu/0.5 mL, wherein based on said totalconcentration the concentration of a dengue serotype 2 (e.g. dengueserotype 2 strain) measured in pfu/0.5 mL is less than 10% of the totalconcentration, or less than 8%, or less than 6% of the totalconcentration, and wherein the concentration of a dengue serotype 4(e.g. chimeric dengue serotype 2/4 strain) measured in pfu/0.5 mL is atleast 50% or at least 60% or at least 65% of the total concentration. Inone embodiment, based on said total concentration the concentration of adengue serotype 2 (e.g. dengue serotype 2 strain) measured in pfu/0.5 mLis 0.3 to 10% or 0.5 to 8% of the total concentration and theconcentration of a dengue serotype 4 (e.g. chimeric dengue serotype 2/4strain) measured in pfu/0.5 mL is 50% to 90% or 60% to 88% of the totalconcentration. This means that the concentration of the dengue serotype2 (e.g. dengue serotype 2 strain) is lower than the concentration of thedengue serotype 4 (e.g. chimeric dengue serotype 2/4 strain).

In one such embodiment, the concentration of a dengue serotype 1 (e.g.chimeric dengue serotype 2/1 strain) measured in pfu/0.5 mL is at least1% of the total concentration, and/or the concentration of a dengueserotype 3 (e.g. chimeric dengue serotype 2/3 strain) measured inpfu/0.5 mL is at least 6% of the total concentration, or at least 7% or8%, 10%, 12%, 14%, 16% or 18% of the total concentration. In one suchembodiment, the concentration of a dengue serotype 2 (e.g. chimericdengue serotype 2/1 strain) measured in pfu/0.5 mL is 1% to 7% or 2% to6% or 2.0% to 5.0% of the total concentration, and/or the concentrationof a dengue serotype 3 (e.g. chimeric dengue serotype 2/3 strain)measured in pfu/0.5 mL is 6% to 25% or 7% to 25% or 10% to 25% or 18% to25% of the total concentration. This means that the concentration of thedengue serotype 1 (e.g. chimeric dengue serotype 2/1 strain) is lowerthan the concentration of the dengue serotype 3 (e.g. chimeric dengueserotype 2/3 strain).

In a preferred embodiment, the concentration of a dengue serotype 2strain, such as TDV-2, measured in pfu/0.5 mL is less than 10% of thetotal concentration, preferably less than 6% or less than 2%, theconcentration of a dengue serotype 4 (e.g. chimeric dengue serotype 2/4strain), such as TDV-4, measured in pfu/0.5 mL is at least 50% of thetotal concentration, preferably at least 65%, the concentration of adengue serotype 1 (e.g. chimeric dengue serotype 2/1 strain), such asTDV-1, measured in pfu/0.5 mL is at least 1% of the total concentration,preferably between 1% and 7% or 2.0% to 5.0%, and the concentration of adengue serotype 3 (e.g. chimeric dengue serotype 2/3 strain), such asTDV-3, measured in pfu/0.5 mL is at least 6% of the total concentration,preferably between 6% and 25% or 10% to 25% or 18% to 25%.

In a further preferred embodiment, a dengue virus composition comprisinga dengue serotype 1 (e.g. chimeric dengue serotype 2/1 strain), a dengueserotype 2 (e.g. dengue serotype 2 strain), a dengue serotype 1 (e.g.chimeric dengue serotype 2/3 strain), and a dengue serotype 4 (e.g.chimeric dengue serotype 2/4 strain), such as TDV-1, TDV-2, TDV-3 andTDV-4, is provided, wherein the concentration of the dengue serotype 1(e.g. chimeric dengue serotype 2/1 strain) measured in pfu/0.5 mL is atleast 1% of the total concentration, preferably between 1% and 7% or2.0% and 5.0%, the concentration of the dengue serotype 2 (e.g. dengueserotype 2 strain) measured in pfu/0.5 mL is less than 10% of the totalconcentration, preferably less than 6% or less than 2% and theconcentration of the dengue serotype 3 (e.g. chimeric dengue serotype2/3 strain) measured in pfu/0.5 mL is at least 6% of the totalconcentration, preferably between 6% and 25% or 10% to 25% or 18% to25%. It is particularly preferred that the dengue serotype 4 (e.g.chimeric dengue serotype 2/4 strain) has the highest concentration ofall four dengue serotypes.

In a further preferred embodiment, the dengue vaccine compositioncomprises a tetravalent dengue virus composition including four liveattenuated dengue virus strains, wherein the concentration of the dengueserotype 1 (e.g. chimeric dengue serotype 2/1 strain) measured inpfu/0.5 mL is 1% to 7% of the total concentration, the concentration ofthe dengue serotype 2 (e.g. dengue serotype 2 strain) measured inpfu/0.5 mL is less than 8% of the total concentration, such as in therange of 1% to 8% of the total concentration, the concentration of thedengue serotype 3 (e.g. chimeric dengue serotype 2/3 strain) measured inpfu/0.5 mL is at least 10% of the total concentration, and theconcentration of the dengue serotype 4 (e.g. chimeric dengue serotype2/4 strain) measured in pfu/0.5 mL is at least 65% of the totalconcentration, such as in the range of 65% to 80%. In certain suchembodiments, the arithmetic sum of all four serotypes is in the range of4.6 log 10 pfu/0.5 mL to 6.7 log 10 pfu/0.5 mL, preferably in the rangeof 4.6 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL.

In a further preferred embodiment the dengue serotype 1 (e.g. chimericdengue serotype 2/1 strain) such as TDV-1 and the dengue serotype 2(e.g. dengue serotype 2 strain) such as TDV-2 are present each in aconcentration based on the total concentration in pfu/0.5 mL which iswithin 5%-points of each other and/or are together less than about 10%of the total concentration in pfu/0.5 mL. In certain such embodimentsthe dengue serotype 3 (e.g. chimeric dengue serotype 2/3 strain) such asTDV-3 is preferably at least about 10% of the total concentration inpfu/0.5 mL and more preferably the dengue serotype 4 (e.g. chimericdengue serotype 2/4 strain) such as TDV-4 is at least about 70% of thetotal concentration in pfu/0.5 mL. In certain such embodiments thedengue serotype 4 (e.g. chimeric dengue serotype 2/4 strain) such asTDV-4 represents the highest concentration in the composition of allfour serotypes, preferably with at least about 70% of the totalconcentration in pfu/0.5 mL, dengue serotype 3 (e.g. chimeric dengueserotype 2/3 strain) such as TDV-3 represents the second highestconcentration in the composition of all four serotypes, preferably withat least about 10% of the total concentration in pfu/0.5 mL, and dengueserotype 1 (e.g. chimeric dengue serotype 2/1 strain) such as TDV-1 anddengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 eachrepresent lower concentrations than the concentration of serotype 3(e.g. chimeric dengue serotype 2/3 strain) such as TDV-3, and optionallytogether represent less than about 10% of the total concentration inpfu/0.5 mL.

Preferably, in said embodiments the chimeric dengue serotype 2/1 strainis TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengueserotype 2/3 strain is TDV-3 and the chimeric dengue serotype 2/4 strainis TDV-4. More preferably, TDV-1 is characterized by the nucleotidesequence according to SEQ ID No. 1 and the amino acid sequence accordingto SEQ ID No. 2, TDV-2 is characterized by the nucleotide sequenceaccording to SEQ ID No. 3 and the amino acid sequence according to SEQID No. 4, TDV-3 is characterized by the nucleotide sequence according toSEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 andTDV-4 is characterized by the nucleotide sequence according to SEQ IDNo. 7 and the amino acid sequence according to SEQ ID No. 8.

According to a further embodiment, the chimeric dengue serotype 2/4strain, preferably TDV-4, has the highest concentration in the denguevaccine composition, followed by the chimeric dengue serotype 2/3strain, preferably TDV-3, followed by the chimeric dengue serotype 2/1strain, preferably TDV-1, followed by the dengue serotype 2 strain,preferably TDV-2. It is particularly preferred that the dengue serotype2 strain has the lowest concentration of the four strains present in thedengue vaccine composition.

Whenever reference is made to a concentration/0.5 ml, this does notlimit the volume of the unit dose described herein to 0.5 ml. 0.5 ml isthe reference volume for the determination of the concentrations of thevirus strains in the composition in pfu/ml. The volume and/or amount perunit dose is described in the respective chapter.

Pharmaceutically Acceptable Excipients

The present invention is in part directed to a unit dose of a denguevaccine composition, wherein the dengue vaccine composition comprisesone or more pharmaceutically acceptable excipients. In one embodiment,the dengue vaccine composition comprises a non-reducing sugar, asurfactant, a protein and an inorganic salt. Preferably, thenon-reducing sugar is trehalose, the surfactant is poloxamer 407, theprotein is human serum albumin and the inorganic salt is sodiumchloride.

In one embodiment, the unit dose of a dengue vaccine compositioncomprises the following pharmaceutically acceptable excipients:

-   -   from about 10% w/v to about 20% w/v α,α-trehalose dihydrate or        an equimolar amount of other forms of α,α-trehalose,    -   from about 0.5% w/v to about 1.5% w/v poloxamer 407,    -   from about 0.05% w/v to about 2% w/v human serum albumin, and    -   from about 70 mM to 140 mM sodium chloride.

In one embodiment, the unit dose of a dengue vaccine compositioncomprises the following pharmaceutically acceptable excipients whenmeasured in 0.5 ml:

-   -   from about 10% w/v to about 20% w/v α,α-trehalose or an        equimolar amount of other forms of α,α-trehalose,    -   from about 0.5% w/v to about 1.5% w/v poloxamer 407,    -   from about 0.05% w/v to about 2% w/v human serum albumin, and    -   from about 70 mM to 140 mM sodium chloride, and preferably    -   has a pH of 7 to 8.5.

In one embodiment, the unit dose of a dengue vaccine compositioncomprises the following pharmaceutically acceptable excipients whenmeasured in 0.5 ml:

-   -   from about 143 mg/ml to about 185 mg/ml α,α-trehalose dihydrate        or an equimolar amount of other forms of α,α-trehalose,    -   from about 9.1 mg/ml to about 12.4 mg/ml poloxamer 407,    -   from about 0.88% mg/ml to about 1.32 mg/ml human serum albumin,        and    -   from about 70 mM to 140 mM sodium chloride, and preferably has a        pH of 7 to 8.5.

In a preferred embodiment, the lyophilized unit dose of the invention asdescribed herein comprises the following pharmaceutically acceptableexcipients:

about 15% w/v α,α-trehalose dihydrate,

-   -   about 1% w/v poloxamer 407,    -   about 0.1% w/v human serum albumin, and    -   about 100 mM sodium chloride.

In a preferred embodiment, the lyophilized unit dose of the invention asdescribed herein comprises the following pharmaceutically acceptableexcipients when measured in 0.5 ml:

-   -   about 15% w/v α,α-trehalose,    -   about 1% w/v poloxamer 407,    -   about 0.1% w/v human serum albumin, and    -   about 100 mM sodium chloride.

In a preferred embodiment, the lyophilized unit dose of the invention asdescribed herein comprises the following pharmaceutically acceptableexcipients:

-   -   about 82.9 mg α,α-trehalose dihydrate,    -   about 5 mg poloxamer 407,    -   about 0.5 mg human serum albumin, and    -   about 50 μmoles sodium chloride.

In a preferred embodiment, the reconstituted unit dose of the inventionas described herein comprises the following pharmaceutically acceptableexcipients:

-   -   about 15% w/v α,α-trehalose dihydrate,    -   about 1% w/v poloxamer 407,    -   about 0.1% w/v human serum albumin, and    -   about 137 mM sodium chloride, and preferably    -   has a pH of 7 to 8.5

In a preferred embodiment, the reconstituted unit dose of the inventionas described herein comprises the following pharmaceutically acceptableexcipients when measured in 0.5 ml:

-   -   about 15% w/v α,α-trehalose,    -   about 1% w/v poloxamer 407,    -   about 0.1% w/v human serum albumin, and preferably    -   about 137 mM sodium chloride and preferably    -   has a pH of 7 to 8.5.

In a preferred embodiment, the reconstituted unit dose of the inventionas described herein comprises the following pharmaceutically acceptableexcipients:

-   -   about 82.9 mg α,α-trehalose dihydrate,    -   about 5 mg poloxamer 407,    -   about 0.5 mg human serum albumin, and preferably    -   about 68.5 μmoles sodium chloride, and preferably    -   has a pH of 7 to 8.5.

The human serum albumin may be a native or recombinant human serumalbumin (rHSA). The poloxamer 407 may be e.g. Pluronic F127.

In one embodiment, the unit dose further comprises a buffer. The buffermay be phosphate buffered saline (PBS). The buffer may include at leastone of sodium chloride (NaCl), monosodium dihydrogen phosphate(NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), potassium chloride(KCl), and potassium dihydrogen phosphate (KH₂PO₄).

In a preferred embodiment, the buffer may include disodium hydrogenphosphate (Na₂HPO₄), potassium chloride (KCl), and potassium dihydrogenphosphate (KH₂PO₄). The buffer may have a pH in the range of 7.0 to 8.5at 25° C.

Unit Dose

The present invention is directed in part to a unit dose of a denguevaccine composition comprising a tetravalent dengue virus composition asdescribed herein and pharmaceutically acceptable excipients as describedherein.

The present invention is directed in part to a unit dose of a denguevaccine composition as described above e.g. of

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) with a concentration of at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) with a        concentration of at least 2.7 log 10 pfu/0.5 mL,    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) with a concentration of at least 4.0 log 10 pfu/0.5 mL,        and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) with a concentration of at least 4.5 log 10 pfu/0.5 mL.

Preferably, the chimeric dengue serotype 2/1 strain is TDV-1, the dengueserotype 2 strain is TDV-2, the chimeric dengue serotype 2/3 strain isTDV-3, and the chimeric dengue serotype 2/4 strain is TDV-4. Morepreferably, TDV-1 is characterized by the nucleotide sequence accordingto SEQ ID No. 1 and the amino acid sequence according to SEQ ID No. 2,TDV-2 is characterized by the nucleotide sequence according to SEQ IDNo. 3 and the amino acid sequence according to SEQ ID No. 4, TDV-3 ischaracterized by the nucleotide sequence according to SEQ ID No. 5 andthe amino acid sequence according to SEQ ID No. 6 and TDV-4 ischaracterized by the nucleotide sequence according to SEQ ID No. 7 andthe amino acid sequence according to SEQ ID No. 8.

In one embodiment, the unit dose is lyophilized. In one such embodiment,the lyophilized unit dose is obtained by subjecting a volume of 0.5 mLof the aqueous dengue vaccine composition produced by combiningpharmaceutically acceptable excipients as described herein and thedengue vaccine composition as described herein comprising the fourdengue virus strains, in particular TDV-1 to TDV-4, to lyophilization.In a preferred embodiment the residual moisture content as determined byKarl Fischer Determination is equal to or less than 5.0%, preferablyequal to or less than 3%.

In another embodiment, the unit dose is reconstituted. The reconstitutedunit dose is obtained by subjecting the lyophilized unit dose toreconstitution with a pharmaceutically acceptable diluent, preferablybefore administration of the dengue vaccine. In one such embodiment,reconstitution will be accomplished by adding a pharmaceuticallyacceptable diluent, such as water for injection, phosphate bufferedsaline or an aqueous sodium chloride solution, to the lyophilized unitdose. In one embodiment, an aqueous sodium chloride solution, such as a37 mM aqueous sodium chloride solution, is added to the lyophilized unitdose for reconstitution. In one such embodiment, the lyophilized unitdose will be reconstituted with 0.3 to 0.8 mL, or 0.4 to 0.7 mL, or 0.5mL of diluent. In a preferred embodiment, the lyophilized unit dose isreconstituted with 0.3 to 0.8 mL, 0.4 to 0.7 mL or 0.5 mL of 37 mMaqueous sodium chloride solution. In a more preferred embodiment, thelyophilized unit dose is reconstituted with 0.5 mL of 37 mM aqueoussodium chloride solution. The reconstituted unit dose can subsequentlybe administered subcutaneously.

It is preferred that the unit dose in lyophilized form is the finalproduct after manufacture of the unit dose and the storage form of theunit dose, wherein the unit dose in reconstituted form is preparedbefore administration of the unit dose to a subject.

The present invention is, moreover, directed in part to a unit dose of adengue vaccine composition comprising:

a tetravalent virus composition including four live attenuated denguevirus strains, wherein the unit dose is lyophilized and uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises:

-   -   (i) a dengue serotype 1, such as a chimeric dengue serotype 2/1        strain, in a concentration of at least 3.3 log 10 pfu/0.5 ml,    -   (ii) a dengue serotype 2, such as a dengue serotype 2 strain, in        a concentration of at least 2.7 log 10 pfu/0.5 ml,    -   (iii) a dengue serotype 3, such as a chimeric dengue serotype        2/3 strain, in a concentration of at least 4.0 log 10 pfu/0.5        ml, and    -   (iv) a dengue serotype 4, such as a chimeric dengue serotype 2/4        strain, in a concentration of at least 4.5 log 10 pfu/0.5 ml.

In one embodiment, the reconstituted unit dose has a volume of e.g. 0.5mL, wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said concentration, the concentration of (iii)at least 10% of the total concentration in pfu/0.5 mL.

In another embodiment the reconstituted unit dose has a volume of e.g.0.5 mL, wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) inpfu/0.5 mL is at least 50%, and the concentration of (i) in pfu/0.5 mLis at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%,or at least 18%.

It is preferred that the concentration in the reconstituted unit dose of(iii) in pfu/0.5 mL is at least 10%.

In one embodiment the reconstituted unit dose has a volume of e.g. 0.5mL, wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is less than 2%, the concentration of (iv) in pfu/0.5mL is at least 50%, the concentration of (i) in pfu/0.5 mL is at least1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%.

In one embodiment, the present invention is directed to a lyophilizedunit dose of a dengue vaccine composition comprising upon reconstitutionwith 0.5 mL of a pharmaceutically acceptable diluent a dengue serotype 1(e.g. chimeric dengue serotype 2/1 strain) with a concentration of atleast 3.3 log 10 pfu/0.5 mL, a dengue serotype 2 (e.g. dengue serotype 2strain) with a concentration of at least 2.7 log 10 pfu/0.5 mL, a dengueserotype 3 (e.g. chimeric dengue serotype 2/3 strain) with aconcentration of at least 4.0 log 10 pfu/0.5 mL, and a dengue serotype 4(e.g. chimeric dengue serotype 2/4 strain) with a concentration of atleast 4.5 log 10 pfu/0.5 mL and pharmaceutically acceptable excipientsas described herein, wherein the unit dose is preferably formulated in0.5 mL before lyophilization. Preferably, the chimeric dengue serotype2/1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimericdengue serotype 2/3 strain is TDV-3 and the chimeric dengue serotype 2/4strain is TDV-4. More preferably, TDV-1 is characterized by thenucleotide sequence according to SEQ ID No. 1 and the amino acidsequence according to SEQ ID No. 2, TDV-2 is characterized by thenucleotide sequence according to SEQ ID No. 3 and the amino acidsequence according to SEQ ID No. 4, TDV-3 is characterized by thenucleotide sequence according to SEQ ID No. 5 and the amino acidsequence according to SEQ ID No. 6 and TDV-4 is characterized by thenucleotide sequence according to SEQ ID No. 7 and the amino acidsequence according to SEQ ID No. 8.

In one such embodiment, the lyophilized unit dose is obtained bylyophilizing 0.5 mL of a dengue vaccine composition comprising a dengueserotype 1 (e.g. chimeric dengue serotype 2/1 strain) in a concentrationof 3.3 log 10 pfu/dose to 5.0 log 10 pfu/0.5 mL, a dengue serotype 2(e.g. dengue serotype 2 strain) in a concentration of 2.7 log 10pfu/dose to 4.9 log 10 pfu/0.5 mL, a dengue serotype 3 (e.g. chimericdengue serotype 2/3 strain) in a concentration of 4.0 log 10 pfu/dose to5.7 log 10 pfu/0.5 mL, and a dengue serotype 4 (e.g. chimeric dengueserotype 2/4 strain) in a concentration of 4.5 log 10 pfu/dose to 5.5log 10 pfu/0.5 mL and pharmaceutically acceptable excipients asdescribed herein. Preferably, the chimeric dengue serotype 2/1 strain isTDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengueserotype 2/3 strain is TDV-3 and the chimeric dengue serotype 2/4 strainis TDV-4.

In one such embodiment, the lyophilized unit dose is obtained bylyophilizing 0.5 mL of a dengue vaccine composition comprising a dengueserotype 1 (e.g. chimeric dengue serotype 2/1 strain) in a concentrationof 3.3 log 10 pfu/0.5 mL to 3.6 log 10 pfu/0.5 mL, a dengue serotype 2(e.g. dengue serotype 2 strain) in a concentration of 2.7 log 10 pfu/0.5mL to 4.0 log 10 pfu/0.5 mL, a dengue serotype 3 (e.g. chimeric dengueserotype 2/3 strain) in a concentration of 4.0 log 10 pfu/0.5 mL to 4.6log 10 pfu/0.5 mL, and a dengue serotype 4 (e.g. chimeric dengueserotype 2/4 strain) in a concentration of 4.5 log 10 pfu/0.5 mL or 4.6log 10 pfu/0.5 mL to 5.1 log 10 pfu/0.5 mL and pharmaceuticallyacceptable excipients as described herein. Preferably, the chimericdengue serotype 2/1 strain is TDV-1, the dengue serotype 2 strain isTDV-2, the chimeric dengue serotype 2/3 strain is TDV-3 and the chimericdengue serotype 2/4 strain is TDV-4.

In certain embodiments, the lyophilized unit dose refers to 0.5 mLbefore lyophilization, wherein TDV-2 and TDV-4 are present in certainrelative amounts, based on the total concentration of TDV-1, TDV-2,TDV-3 and TDV-4 in pfu/0.5 mL, and the concentration of TDV-2 measuredin pfu/0.5 mL is less than 10% or less than 8% or less than 6%, and theconcentration of TDV-4 measured in pfu/0.5 mL is at least 50% or atleast 65%. In some of these embodiments, the concentration of TDV-1measured in pfu/0.5 mL is at least 1% and/or the concentration of TDV-3measured in pfu/0.5 mL is at least 6%, 7%, 8%, 10%, 12%, 14%, 16% k orat least 18%.

In certain embodiments, the reconstituted unit dose has a volume of 0.5mL and TDV-2 and TDV-4 are present in certain relative amounts, based onthe total concentration of TDV-1, TDV-2, TDV-3 and TDV-4 in pfu/0.5 mL,and the concentration of TDV-2 measured in pfu/0.5 mL is less than 10%or less than 8% or less than 6%, and the concentration of TDV-4 measuredin pfu/0.5 mL is at least 50% or at least 65%. In some of theseembodiments, the concentration of TDV-1 measured in pfu/0.5 mL is atleast 1% and/or the concentration of TDV-3 measured in pfu/0.5 mL is atleast 6%, 7%, 8%, 10%, 12%, 14%, 16% k or at least 18%.

In a further preferred embodiment, the reconstituted unit dose has avolume of 0.5 mL and comprises a tetravalent dengue virus compositionincluding four live attenuated dengue virus strains, wherein theconcentration of the dengue serotype 1 (e.g. dengue serotype 2/1 strain)measured in pfu/0.5 mL is 1% to 7% of the total concentration, theconcentration of the dengue serotype 2 (e.g dengue serotype 2 strain)measured in pfu/0.5 mL is less than 8% of the total concentration, suchas in the range of 1% to 8% of the total concentration, theconcentration of the dengue serotype 3 (e.g. dengue serotype 2/3 strain)measured in pfu/0.5 mL is at least 10% of the total concentration, andthe concentration of the dengue serotype 4 (e.g. dengue serotype 2/4strain) measured in pfu/0.5 mL is at least 65% of the totalconcentration, such as in the range of 65% to 80%. In certain suchembodiments, the arithmetic sum of all four serotypes is in the range of4.6 log 10 pfu/0.5 mL to 6.7 log 10 pfu/0.5 mL, preferably in the rangeof 4.6 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL.

In a further preferred embodiment, the reconstituted unit dose has avolume of 0.5 mL and comprises a tetravalent dengue virus compositionincluding four live attenuated dengue virus strains, wherein the dengueserotype 1 (e.g. chimeric dengue serotype 2/1 strain) such as TDV-1 andthe dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 arepresent each in a concentration based on the total concentration inpfu/0.5 mL which is within 5%-points of each other and/or are togetherless than about 10% of the total concentration in pfu/0.5 mL. In certainsuch embodiments the dengue serotype 3 (e.g. chimeric dengue serotype2/3 strain) such as TDV-3 is preferably at least about 10% of the totalconcentration in pfu/0.5 mL and more preferably the dengue serotype 4(e.g. chimeric dengue serotype 2/4 strain) such as TDV-4 is at leastabout 70% of the total concentration in pfu/0.5 mL. In certain suchembodiments the dengue serotype 4 (e.g. chimeric dengue serotype 2/4strain) such as TDV-4 represents the highest concentration in thecomposition of all four serotypes, preferably with at least about 70% ofthe total concentration in pfu/0.5 mL, dengue serotype 3 (e.g. chimericdengue serotype 2/3 strain) such as TDV-3 represents the second highestconcentration in the composition of all four serotypes, preferably withat least about 10% of the total concentration in pfu/0.5 mL, and dengueserotype 1 (e.g. chimeric dengue serotype 2/1 strain) such as TDV-1 anddengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 eachrepresent lower concentrations than the concentration of serotype 3(e.g. chimeric dengue serotype 2/3 strain) such as TDV-3, and optionallytogether represent less than about 10% of the total concentration inpfu/0.5 mL.

The lyophilized unit dose reconstituted in 0.5 mL will provide the aboveconcentrations for the four dengue serotypes. While the unit dose of adengue vaccine composition as described herein refers to theconcentrations of the dengue serotypes in 0.5 mL, the lyophilized unitdose can be reconstituted with other volumes of a pharmaceuticallyacceptable diluent, such as an aqueous sodium chloride solution, withoutchanging the absolute virus amount administered or the ratios of theviruses to one another.

In certain embodiments, the lyophilized unit dose of the invention isprepared from a solution comprising a non-reducing sugar, a surfactant,a protein and an inorganic salt.

In certain embodiments, the lyophilized unit dose of the invention isprepared from a solution comprising trehalose, poloxamer 407, humanserum albumin and sodium chloride.

In certain embodiments, the lyophilized unit dose of the invention isprepared from a solution comprising about 10% w/v to about 20% w/vα,α-trehalose dihydrate or an equimolar amount of other forms ofα,α-trehalose, from about 0.5% w/v to about 1.5% w/v poloxamer 407, fromabout 0.05% w/v to about 2% w/v human serum albumin, and about 70 mM toabout 120 mM sodium chloride.

In preferred embodiments, the lyophilized unit dose of the invention asdescribed herein is prepared from a solution comprising about 15% w/vα,α-trehalose dihydrate, about 1% w/v poloxamer 407, about 0.1% w/vhuman serum albumin and about 100 mM sodium chloride.

In one embodiment, the solution from which the lyophilized unit dose isprepared further comprises a buffer. The buffer may be phosphatebuffered saline (PBS). The buffer may include at least one of sodiumchloride (NaCl), monosodium dihydrogen phosphate (NaH₂PO₄), disodiumhydrogen phosphate (Na₂HPO₄), potassium chloride (KCl), and potassiumdihydrogen phosphate (KH₂PO₄). In a preferred embodiment, the buffer mayinclude disodium hydrogen phosphate (Na₂HPO₄), potassium chloride (KCl),and potassium dihydrogen phosphate (KH₂PO₄). The buffer may have a pH inthe range of about 7.0 to about 8.5 at 25° C. or a pH of about 6.8 toabout 7.6 at 25° C., preferably a pH of about 7.2 at 25° C.

In preferred embodiments, the reconstituted unit dose of the inventionas described herein comprising about 15% w/v α,α-trehalose dihydrate,about 1% w/v poloxamer 407, about 0.1% w/v human serum albumin and about137 mM sodium chloride. The reconstituted unit dose may have a pH ofabout 7.0 to about 8.5 at 25° C., preferably a pH of about 7.2 at 25° C.

The unit dose of the invention as described herein activates multiplearms of the immune system—neutralizing antibodies, cellular immunity andanti-NS1 antibodies—in both seronegative and seropositive subjectpopulations or in both seronegative and seropositive subjects. Thus, theunit dose of the invention as described herein protects both dengueseronegative and dengue seropositive subject populations or subjectsagainst dengue disease.

In one embodiment, one unit dose is present in a container, preferably avial, and said unit dose is administered to a subject afterreconstitution. In one embodiment, more than one unit dose of the denguevaccine composition may be present in a container, preferably a vial, sothat with the content of one container, preferably a vial, more than onesubject can be vaccinated. In one embodiment, the container comprisingmore than one unit doses of the invention as described herein is usedfor providing the reconstituted unit dose to be used in the methods ofthe invention as described herein.

The certain embodiments, the container comprising the unit dose of theinvention is part of a kit. Thus, the invention is directed in part to akit for preparing a reconstituted unit dose comprising a lyophilizedunit dose of the present invention as described herein, and apharmaceutically acceptable diluent for reconstitution.

In certain embodiments, the diluent for reconstitution provided in acontainer, preferably a vial, or a pre-filled syringe. In someembodiments, the diluent for reconstitution is selected from water forinjection, phosphate buffered saline or an aqueous sodium chloridesolution. In a preferred embodiment, the diluent for reconstitution is30 to 40 mM sodium chloride, such as 37 mM sodium chloride.

In certain embodiments, the kit may further comprise a yellow fevervaccine, in particular YF-17D. In some embodiments, the yellow fevervaccine may be in a separate container, such as a vial. In anotherembodiment, the yellow fever vaccine and the unit dose of the inventionmay be in the same container. Thus, the invention is directed in part toa combined dengue/yellow fever vaccine, wherein the unit dose of theinvention as described herein is combined with a yellow fever vaccine.Such a combined dengue/yellow fever vaccine comprises the unit dose ofthe invention as described herein and a yellow fever vaccine, inparticular YF-17D, in the same formulation. In certain embodiments, theinvention is directed to a kit comprising such a combined dengue/yellowfever vaccine and a unit dose of the invention as described herein.

In certain embodiments, the kit may further comprise a hepatitis Avaccine, such as HAVRIX® or VAQTA®. In some embodiments, the hepatitis Avaccine may be in a separate container, such as a vial. In anotherembodiment, the hepatitis A vaccine and the unit dose of the inventionmay be in the same container. Thus, the invention is directed in part toa combined dengue/hepatitis A vaccine, wherein the unit dose of theinvention as described herein is combined with a hepatitis A vaccine.Such a combined dengue/hepatitis A vaccine comprises the unit dose ofthe invention as described herein and a hepatitis A vaccine, such asHAVRIX® or VAQTA®, in the same formulation. In certain embodiments, theinvention is directed to a kit comprising such a combineddengue/hepatitis A vaccine and a unit dose of the invention as describedherein.

In certain embodiments, the kit may further comprise a HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9. In some embodiments,the HPV vaccine may be in a separate container, such as a vial. Inanother embodiment, the HPV vaccine and the unit dose of the inventionmay be in the same container. Thus, the invention is directed in part toa combined dengue/HPV vaccine, wherein the unit dose of the invention asdescribed herein is combined with a HPV vaccine. Such a combineddengue/HPV vaccine comprises the unit dose of the invention as describedherein and a HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9, in the same formulation. In certain embodiments, theinvention is directed to a kit comprising such a combined dengue/HPVvaccine and a unit dose of the invention as described herein.

In certain embodiments, the kit may further comprise a MMR vaccine, suchas M-M-R® II. In some embodiments, the MMR vaccine may be in a separatecontainer, such as a vial. In another embodiment, the MMR vaccine andthe unit dose of the invention may be in the same container. Thus, theinvention is directed in part to a combined dengue/MMR vaccine, whereinthe unit dose of the invention as described herein is combined with aMMR vaccine. Such a combined dengue/MMR vaccine comprises the unit doseof the invention as described herein and a MMR vaccine, such as M-M-R®II, in the same formulation. In certain embodiments, the invention isdirected to a kit comprising such a combined dengue/MMR vaccine and aunit dose of the invention as described herein.

In certain embodiments, the kit may further comprise a Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®. In someembodiments, the Tdap vaccine may be in a separate container, such as avial. In another embodiment, the Tdap vaccine and the unit dose of theinvention may be in the same container. Thus, the invention is directedin part to a combined dengue/Tdap vaccine, wherein the unit dose of theinvention as described herein is combined with a Tdap vaccine. Such acombined dengue/Tdap vaccine comprises the unit dose of the invention asdescribed herein and a Tdap vaccine, in particular a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, in the same formulation. In certainembodiments, the invention is directed to a kit comprising such acombined dengue/Tdap vaccine and a unit dose of the invention asdescribed herein.

In certain embodiments, the kit may further comprise a DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®. In some embodiments, the DTaP/IPV/Hib vaccine may be in aseparate container, such as a vial. In another embodiment, theDTaP/IPV/Hib vaccine and the unit dose of the invention may be in thesame container. Thus, the invention is directed in part to a combineddengue/DTaP/IPV/Hib vaccine, wherein the unit dose of the invention asdescribed herein is combined with a DTaP/IPV/Hib vaccine. Such acombined dengue/DTaP/IPV/Hib vaccine comprises the unit dose of theinvention as described herein and a DTaP/IPV/Hib vaccine, in particulara combined DTaP/IPV/Hib vaccine, such as Pentacel®, in the sameformulation. In certain embodiments, the invention is directed to a kitcomprising such a combined dengue/DTaP/IPV/Hib vaccine and a unit doseof the invention as described herein.

Yellow Fever Vaccine

YF-VAX®, a yellow fever vaccine from Sanofi, for subcutaneous use, isprepared by culturing the YF-17D strain of yellow fever virus in livingavian leukosis virus-free (ALV-free) chicken embryos. The vaccinecontains sorbitol and gelatin as a stabilizer and is lyophilized. Nopreservative is added. YF-VAX is formulated to contain not less than4.74 log₁₀ pfu per 0.5 mL dose throughout the life of the product.

Hepatitis A Vaccine

HAVRIX®, a hepatitis A vaccine from GlaxoSmithKline, is a sterilesuspension of inactivated virus for intramuscular administration. Thevirus (strain HM175) is propagated in MRC-5 human diploid cells. Afterremoval of the cell culture medium, the cells are lysed to form asuspension. This suspension is purified through ultrafiltration and gelpermeation chromatography procedures. Treatment of this lysate withformalin ensures viral inactivation. Viral antigen activity isreferenced to a standard using an enzyme linked immunosorbent assay(ELISA), and is therefore expressed in terms of ELISA Units (EL.U.).Each 1-mL dose for adults 18 years of age) of vaccine contains 1440EL.U. of viral antigen, adsorbed on 0.5 mg of aluminum as aluminumhydroxide. Each 0.5-mL dose for children and adolescents (12 monthsthrough 18 years of age) of vaccine contains 720 EL.U. of viral antigen,adsorbed onto 0.25 mg of aluminum as aluminum hydroxide. HAVRIX®contains the following excipients: Amino acid supplement (0.3% w/v) in aphosphate-buffered saline solution and polysorbate 20 (0.05 mg/mL). Fromthe manufacturing process, HAVRIX® also contains residual MRC-5 cellularproteins (not more than 5 μg/mL), formalin (not more than 0.1 mg/mL),and neomycin sulfate (not more than 40 ng/mL), an aminoglycosideantibiotic included in the cell growth media. HAVRIX® is formulatedwithout preservatives.

VAQTA®, a hepatitis A vaccine from Merck Sharp & Dohme Corp., is aninactivated whole virus vaccine derived from hepatitis A virus grown incell culture in human MRC-5 diploid fibroblasts. It contains inactivatedvirus of a strain, which was originally derived by further serialpassage of a proven attenuated strain. The virus is grown, harvested,purified by a combination of physical and high performance liquidchromatographic techniques developed at the Merck Research Laboratories,formalin inactivated, and then adsorbed onto amorphous aluminumhydroxyphosphate sulfate. VAQTA® is a sterile suspension forintramuscular injection. One milliliter of the vaccine containsapproximately 50 U of hepatitis A virus antigen, which is purified andformulated without a preservative. Within the limits of current assayvariability, the 50 U dose of VAQTA® contains less than 0.1 μg ofnon-viral protein, less than 4×10⁻⁶ μg of DNA, less than 10⁻⁴ μg ofbovine albumin, and less than 0.8 μg of formaldehyde. Other processchemical residuals are less than 10 parts per billion (ppb), includingneomycin. Each 0.5-mL pediatric dose contains 25 U of hepatitis A virusantigen and adsorbed onto approximately 0.225 mg of aluminum provided asamorphous aluminum hydroxyphosphate sulfate, and 35 μg of sodium borateas a pH stabilizer, in 0.9% sodium chloride. Each 1-mL adult dosecontains 50 U of hepatitis A virus antigen and adsorbed ontoapproximately 0.45 mg of aluminum provided as amorphous aluminumhydroxyphosphate sulfate, and 70 μg of sodium borate as a pH stabilizer,in 0.9% sodium chloride.

HPV Vaccine

GARDASIL® 9, a HPV vaccine from Merck, is a non-infectious recombinant9-valent vaccine prepared from the purified virus-like particles (VLPs)of the major capsid (L1) protein of HPV serotypes 6, 11, 16, 18, 31, 33,45, 52, and 58. The L1 proteins are produced by separate fermentationsusing recombinant Saccharomyces cerevisiae and self-assembled into VLPs.The fermentation process involves growth of S. cerevisiae onchemically-defined fermentation media which include vitamins, aminoacids, mineral salts, and carbohydrates. The VLPs are released from theyeast cells by cell disruption and purified by a series of chemical andphysical methods. The purified VLPs are adsorbed on preformedaluminum-containing adjuvant (amorphous aluminum hydroxyphosphatesulfate or AAHS). The 9-valent HPV VLP vaccine is a sterile liquidsuspension that is prepared by combining the adsorbed VLPs of each HPVserotype and additional amounts of the aluminum-containing adjuvant andthe final purification buffer. GARDASIL 9 is a sterile suspension forintramuscular administration. Each 0.5-mL dose contains approximately 30μg of HPV serotype 6 L1 protein, 40 μg of HPV serotype 11 L1 protein, 60μg of HPV serotype 16 L1 protein, 40 μg of HPV serotype 18 L1 protein,20 μg of HPV serotype 31 L1 protein, 20 μg of HPV serotype 33 L1protein, 20 μg of HPV serotype 45 L1 protein, 20 μg of HPV serotype 52L1 protein, and 20 μg of HPV serotype 58 L1 protein. Each 0.5-mL dose ofthe vaccine also contains approximately 500 μg of aluminum (provided asAAHS), 9.56 mg of sodium chloride, 0.78 mg of L-histidine, 50 μg ofpolysorbate 80, 35 μg of sodium borate.

MMR Vaccine

Several MMR vaccines are known in the prior art and include M-M-R® II,Priorix®, Tresivac®, and Trimovax®.

M-M-R® II, a MMR vaccine from Merck Sharp & Dohme Corp, is a live virusvaccine for vaccination against measles, mumps and rubella. M-M-R® II isa sterile lyophilized preparation of (1) ATTENUVAX® (measles virusvaccine live), a more attenuated line of measles virus, derived fromEnders' attenuated Edmonston strain and propagated in chick embryo cellculture, (2) MUMPSVAX® (mumps virus vaccine live), the Jeryl Lynn™ (Blevel) strain of mumps virus propagated in chick embryo cell culture,and (3) MERUVAX® II (rubella virus vaccine live), the Wistar RA 27/3strain of live attenuated rubella virus propagated in WI-38 humandiploid lung fibroblasts. The growth medium for measles and mumps isMedium 199 (a buffered salt solution containing vitamins and amino acidsand supplemented with fetal bovine serum) containing SPGA (sucrose,phosphate, glutamate, and recombinant human albumin) as stabilizer andneomycin. The growth medium for rubella is Minimum Essential Medium(MEM) (a buffered salt solution containing vitamins and amino acids andsupplemented with fetal bovine serum) containing recombinant humanalbumin and neomycin. Sorbitol and hydrolyzed gelatin stabilizer areadded to the individual virus harvests. The cells, virus pools, andfetal bovine serum are all screened for the absence of adventitiousagents. The reconstituted vaccine is for subcutaneous administration.Each 0.5 mL dose contains not less than 1,000 TCID₅₀ (tissue cultureinfectious doses) of measles virus, 12,500 TCID₅₀ of mumps virus, and1,000 TCID₅₀ of rubella virus. Each dose of the vaccine is calculated tocontain sorbitol (14.5 mg), sodium phosphate, sucrose (1.9 mg), sodiumchloride, hydrolyzed gelatin (14.5 mg), recombinant human albumin (≤0.3mg), fetal bovine serum (<1 ppm), other buffer and media ingredients andapproximately 25 μg of neomycin. The product contains no preservative.The lyophilized vaccine is reconstituted before administration.

Combined Tetanus, Diphtheria, and Pertussis (Tdap) Vaccine

BOOSTRIX®, a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) (Tdap) vaccine, is a non-infectious,sterile vaccine for intramuscular administration. It contains tetanustoxoid, diphtheria toxoid, and pertussis antigens (inactivated pertussistoxin (iPT) and formaldehyde-treated filamentous hemagglutinin (FHA) andpertactin (PRN)). The antigens are the same as those in INFANRIX®, butBOOSTRIX® is formulated with reduced quantities of these antigens.

BOOSTRIX® is supplied as a 0.5 mL suspension for injection. Each 0.5 mLdose of BOOSTRIX® is formulated to contain 5 Limits of flocculation (Lf)of tetanus toxoid, 2.5 Lf of diphtheria toxoid, 8 μg of iPT, 8 μg offormaldehyde treated FHA, and 2.5 μg of PRN (69 kiloDalton outermembrane protein), aluminum hydroxide as adjuvant (not more than 0.39 mgaluminum by assay), 4.5 mg of sodium chloride, 1.00 μg of residualformaldehyde, and 1.00 μg of polysorbate 80 (Tween 80).

Tetanus toxin is produced by growing Clostridium tetani in a modifiedLatham medium derived from bovine casein. The diphtheria toxin isproduced by growing Corynebacterium diphtheriae in Fenton mediumcontaining a bovine extract. Both toxins are detoxified withformaldehyde, concentrated by ultrafiltration, and purified byprecipitation, dialysis, and sterile filtration. Tetanus and diphtheriatoxoid potency is determined by measuring the amount of neutralizingantitoxin in previously immunized guinea pigs.

The acellular pertussis antigens (iPT, FHA, and PRN) are isolated fromBordetella pertussis culture grown in modified Stainer-Scholte liquidmedium. iPT and FHA are isolated from the fermentation broth, PRN isextracted from the cells by heat treatment and flocculation. Theantigens are purified in successive chromatographic and precipitationsteps. iPT is detoxified using glutaraldehyde and formaldehyde. FHA andPRN are treated with formaldehyde. Each antigen is individually adsorbedonto aluminum hydroxide. The potency of the acellular pertussiscomponents (inactivated iPT and formaldehyde-treated FHA and PRN) isdetermined by enzyme-linked immunosorbent assay (ELISA) on sera frompreviously immunized mice.

Combined Diphtheria, Tetanus, Pertussis, Poliomyelitis and Haemophilusinfluenzae Type b (DTaP/IPV/Hib) Vaccine

Pentacel®, a combined DTaP/IPV/Hib vaccine, consists of a diphtheria andtetanus toxoids and acellular pertussis adsorbed and inactivatedpoliovirus (DTaP-IPV) component and an ActHIB® component combinedthrough reconstitution for intramuscular injection. ActHIB® (Haemophilusb Conjugate Vaccine (Tetanus Toxoid Conjugate)), consists ofHaemophilius influenzae type b capsular polysaccharide(polyribosyl-ribitol-phosphate (PRP)) covalently bound to tetanus toxoid(PRP-T). The DTaP-IPV component is supplied as a sterile liquid used toreconstitute the lyophilized ActHIB® component to form Pentacel®.

Each 0.5 mL dose contains 15 Limits of flocculation (Lf) diphtheriatoxoid, 5 Lf tetanus toxoid, acellular pertussis antigens (20 μgdetoxified pertussis toxin (PT), 20 μg filamentous hemagglutinin (FHA),3 μg pertactin (PRN), 5 μg fimbriae types 2 and 3 (FIM)), inactivatedpolioviruses (40 D-antigen units (DU) Type 1 (Mahoney), 8 DU Type 2(MEF-1), 32 DU Type 3 (Saukett)) and 10 μg PRP of Haemophiliusinfluenzae type b covalently bound to 24 μg of tetanus toxoid (PRP-T).

Other ingredients per 0.5 mL dose include 1.5 mg aluminum phosphate(0.33 mg aluminum) as the adjuvant, polysorbate 80 (approximately 10 ppmby calculation), 42.5 mg sucrose, ≤5 μg residual formaldehyde, <50 ngresidual glutaraldehyde, ≤50 ng residual bovine serum albumin, 3.3 mg(0.6% v/v) 2-phenoxyethanol (not as a preservative), <4 pg of neomycinand <4 pg polymyxin B sulfate.

Corynebacterium diphtheriae is grown in modified Mueller's growthmedium. After purification by ammonium sulfate fractionation, thediphtheria toxin is detoxified with formaldehyde and diafiltered.

Clostridium tetani is grown in modified Mueller-Miller casamino acidmedium without beef heart infusion. Tetanus toxin is detoxified withformaldehyde and purified by ammonium sulfate fractionation anddiafiltration.

Diphtheria and tetanus toxoids are individually adsorbed onto aluminumphosphate.

The acellular pertussis vaccine antigens are produced from Bordetellapertussis cultures grown in Stainer-Scholte medium modified by theaddition of casamino acids and dimethyl-beta-cyclodextrin. PT, FHA andPRN are isolated separately from the supernatant culture medium. FIM areextracted and co-purified from the bacterial cells. The pertussisantigens are purified by sequential filtration, salt-precipitation,ultrafiltration and chromatography. PT is detoxified withglutaraldehyde. FHA is treated with formaldehyde and the residualaldehydes are removed by ultrafiltration. The individual antigens areadsorbed separately onto aluminum phosphate.

Poliovirus Type 1, Type 2 and Type 3 are each grown in separate culturesof MRC-5 cells, a line of normal human diploid cells, by themicrocarrier method. The cells are grown in CMRL (Connaught MedicalResearch Laboratories) 1969 medium, supplemented with calf serum. Forviral growth, the culture medium is replaced by Medium 199, without calfserum. After clarification and filtration, the viral suspensions areconcentrated by ultrafiltration, and purified by liquid chromatographysteps. The monovalent viral suspensions are inactivated withformaldehyde. Monovalent concentrates of each inactivated poliovirus arecombined to produce a trivalent poliovirus concentrate.

The adsorbed diphtheria, tetanus and acellular pertussis antigens arecombined with aluminum phosphate (as adjuvant), 2-phenoxyethanol (not asa preservative) and water for injection, into an intermediateconcentrate. The trivalent poliovirus concentrate is added and theDTaP-IPV component is diluted to its final concentration. The DTaP-IPVcomponent does not contain a preservative.

Both diphtheria and tetanus toxoids induce at least 2 neutralizing unitsper mL in the guinea pig potency test. The potency of the acellularpertussis antigens is evaluated by the antibody response of immunizedmice to detoxified PT, FHA, PRN and FIM as measured by enzyme-linkedimmunosorbent assay (ELISA). The potency of inactivated poliovirusantigens is determined by measuring antibody-mediated neutralization ofpoliovirus in sera from immunized rats.

PRP, a high molecular weight polymer, is prepared from the Haemophilusinfluenzae type b strain 1482 grown in a semi-synthetic medium. Thetetanus toxoid for conjugation to PRP is prepared by ammonium sulfatepurification, and formalin inactivation of the toxin from cultures ofClostridium tetani (Harvard strain) grown in a modified Mueller andMiller medium. The toxoid is filter sterilized prior to the conjugationprocess. The ActHIB® component does not contain a preservative. Potencyof the ActHIB® component is specified on each lot by limits on thecontent of PRP polysaccharide and protein per dose and the proportion ofpolysaccharide and protein that is characterized as high molecularweight conjugate.

Method of Preventing and Uses, Method of Inoculating and Uses Method ofPreventing, Method of Inoculating

The present invention is directed in part to a method of preventingdengue disease (in particular virologically confirmable dengue, VCD) ina subject. Thus, in certain embodiments the invention is directed to amethod of preventing dengue disease in a subject, comprisingadministering to the subject, a unit dose/tetravalent dengue viruscomposition, in particular a reconstituted unit dose of the invention asdescribed herein.

The present invention is directed in part to a method of preventingdengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thus, incertain embodiments the invention is directed to a method of preventingdengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS),comprising administering to the subject a reconstituted unitdose/tetravalent dengue virus composition of the invention as describedherein.

The present invention is therefore directed to a method of inoculating asubject against virologically confirmable dengue disease with atetravalent dengue virus composition including four live attenuateddengue virus strains representing serotype 1, serotype 2, serotype 3 andserotype 4, wherein in particular the tetravalent dengue viruscomposition includes a chimeric dengue serotype 2/1 strain and a dengueserotype 2 strain and a chimeric dengue serotype 2/3 strain and achimeric dengue serotype 2/4 strain, wherein in particular the dengueserotype 2 strain is derived from the wild type virus strain DEN-2 16681(SEQ ID NO 11) and differs in at least three nucleotides from the wildtype as follows:

a) 5′-noncoding region (NCR)-57 (nt-57 C-to-T): major attenuation locus

b) NS1-53 Gly-to-Asp (nt-2579 G-to-A): major attenuation locus

c) NS3-250 Glu-to-Val (nt-5270 A-to-T): major attenuation locus; and

wherein the three chimeric dengue strains are derived from the serotype2 strain by replacing the structural proteins prM and E from serotype 2strain with the corresponding structural proteins from the other dengueserotypes, resulting in the following chimeric dengue strains:

a DENV-2/1 chimera,

a DENV-2/3 chimera and

a DENV-2/4 chimera.

Further information regarding the serotypes of the tetravalentcomposition can be derived from section “Dengue virus strains” above.

The tetravalent dengue virus composition for such a method may be in theform of a unit dose comprising:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The present invention is in particular directed to such a method whereinthe unit dose is lyophilized and upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent comprises:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The present invention is also in particular directed to such a methodwherein upon reconstitution with a pharmaceutically acceptable diluent(i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mLand based on said total concentration the concentration of (ii) inpfu/0.5 mL is less than 10%, and the concentration of (iv) in pfu/0.5 mLis at least 50%, and the concentration of (i) in pfu/0.5 mL is at least1%, and the concentration of (iii) in pfu/0.5 mL is at least 8%, or atleast 10%, or at least 12%, or at least 14%, or at least 16%, or atleast 18%, and wherein preferably the subject is 2 to 17 years of age or4 to 16 years of age.

The present invention is also in particular directed to such a methodwherein upon reconstitution with a pharmaceutically acceptable diluent(i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mLand based on said total concentration the concentration of (ii) inpfu/0.5 mL is less than 2%, the concentration of (iv) in pfu/0.5 mL isat least 50%, the concentration of (i) in pfu/0.5 mL is at least 1%, andthe concentration of (iii) in pfu/0.5 mL is at least 6%, whereinpreferably the subject is 18 to 60 years of age.

Further information regarding the tetravalent composition or the unitdose can be derived from section “Dengue vaccine composition” and “Unitdose” above.

The present invention is therefore directed to a method andcorresponding use, the method comprising a primary vaccination with onlytwo administrations of the unit dose comprising the steps of:

-   (A) administering a first unit dose of the tetravalent dengue virus    composition to the subject, and-   (B) administering a second unit does of the tetravalent dengue virus    composition to the subject within 3 months of administration of the    first unit dose.    According to this embodiment the administration of only two doses    within 3 months is sufficient to provide effective protection    against a subsequent dengue infection.

Such method preferably provides a combined vaccine efficacy against allfour serotypes in preventing virologically confirmable dengue diseasewith a 2-sided 95% confidence interval, wherein the lower bound is morethan 60%, when measured against placebo in a subject population of atleast 5,000 healthy subjects irrespective of serostatus at baseline and14 to 16 years of age, from the first administration of theadministration schedule until 18 months after the second administrationof the administration schedule.

Such method also preferably provides a combined vaccine efficacy againstall four serotypes, in preventing virologically confirmable denguedisease with a 2-sided 95% confidence interval, wherein the lower boundis more than 45%, when measured against placebo in a subject populationof at least 1,500 or at least 2,000 healthy subjects seronegativeagainst all serotypes at baseline and 14 to 16 years of age, from 30days after the second administration of the administration scheduleuntil 18 months after the second administration of the administrationschedule.

According to certain embodiments the method of inoculation against thevirologically confirmable dengue disease is due to a dengue serotype 2,and/or due to a dengue serotype 1. The method has very high efficacyagainst dengue serotype 2 and dengue serotype 1 and the highest efficacyagainst dengue serotype 2.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 1, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age from 30 days post second administration until 12 to 18months (e.g. at 12 or at 18 months) after the second administration. Incertain such embodiments, the lower bound is more than 30%, is more than35% is more than 40%, is more than 45%, is more than 50%, or is morethan 54%. In certain such embodiments the subject population of at least1,500 is seronegative against all serotypes at base line and the lowerbound is more than 35%. In certain such embodiments the seronegative andseropositive population each provide a vaccine efficacy against serotype1 with a 2-sided 95% confidence interval, wherein the lower bounds arewithin 10%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 1, in preventing virologicallyconfirmable dengue disease, when measured against placebo in a subjectpopulation of at least 1,500, or at least 2,000, or at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage from 30 days post second administration until 12 to 18 months (e.g.at 12 or at 18 months) after the second administration. In certain suchembodiments, the vaccine efficacy is more than 40%, is more than 50%, ismore than 60%, or is more than 65%. In certain such embodiments thesubject population of at least 1,500 is seronegative against allserotypes at base line. In certain such embodiments the seronegative andseropositive population each provide a vaccine efficacy against serotype1 which are within 5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 2, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age from 30 days post second administration until 12 to 18months (e.g. at 12 or at 18 months) after the second administration. Incertain such embodiments, the lower bound is more than 50%, is more than60%, is more than 70%, is more than 80%, or is more than 85%. In certainsuch embodiments the subject population of at least 1,500, isseronegative against all serotypes. In certain such embodiments theseronegative and seropositive population each provide a vaccine efficacyagainst serotype 2 with a 2-sided 95% confidence interval, wherein thelower bounds are within 5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 2, in preventing virologicallyconfirmable dengue disease, when measured against placebo in a subjectpopulation of at least 1,500, or at least 2,000, or at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage from 30 days post second administration until 12 to 18 months (e.g.at 12 or at 18 months) after the second administration. In certain suchembodiments, the vaccine efficacy is more than 60%, is more than 70%, ismore than 80%, or is more than 90%. In certain such embodiments thesubject population of at least 1,500 is seronegative against allserotypes at base line. In certain such embodiments the seronegative andseropositive population each provide a vaccine efficacy against serotype2 which are within 5%-points.

The efficacy of the method is further described in more detail below inthis the section.

In certain embodiments the unit dose is reconstituted and administeredby subcutaneous injection. According to some of these embodiments, thesubcutaneous injection is administered to the arm, preferably to thedeltoid region of the arm.

According to one embodiment such a method does not include a step ofdetermination whether there was a previous dengue infection in thesubject before administration of the unit dose or wherein the serostatusof the subject is unknown before administration of the unit dose.

According to one embodiment such a method does not include a step ofdetermination of a previous dengue infection in the subjects preferablyat any time before, during or after the steps of administration orwherein the serostatus of the subject is unknown preferably at any timebefore, during or after the steps of administration.

The method according to the invention does not require the testing ofthe serostatus before vaccination and thus allows immediate treatmentand outbreak control. According to certain embodiments the use is for amethod wherein the subject is exposed to a dengue outbreak. In certainsuch embodiments the outbreak is due to a dengue serotype 2, and/or dueto a serotype 1.

According to one embodiment such a method the subject is from a regionwherein the seroprevalence rate is unknown and/or wherein theseroprevalence rate is below 80%, or below 70%, or below 60%.

According to one embodiment of such a method the subject is seronegativeat baseline and is from a region or travels to a region wherein theseroprevalence rate is high with respect to serotype 1 and/or serotype 2i.e. 80%, or 90% or above.

According this embodiment the vaccine and corresponding method is safefor seronegative and seropositive subjects and thus does not require ananalysis of the serostatus or a determination of a previous dengueinfection or a high seroprevalence rate in the region. Such a methodpreferably provides a combined vaccine efficacy againstvirologically-confirmed dengue with hospitalization against all fourserotypes with a 2-sided 95% confidence interval, wherein the lowerbound is more than 65%, when measured against placebo in a subjectpopulation of at least 5,000 healthy 4 to 16 year old subjectsirrespective of serostatus at baseline, preferably in at least 1,500healthy 4 to 16 year old subjects seronegative at baseline, from firstadministration of the administration schedule until 12 to 18 monthsafter the second administration of the administration schedule.Preferably, the 2-sided 95% confidence interval of the combined vaccineefficacy against virologically-confirmed dengue with hospitalizationagainst all four serotypes when comparing seropositive and seronegativesubjects provides for lower bounds of the 2-sided confidence intervalwhich are within 10% points or within 15% points or within 20% points.The method is preferably safe with respect to serotype 1 and serotype 2which may therefore be used in outbreak situations due to serotype 1and/or serotype 2 or even for seronegative subjects (e.g. travelers) orsubjects with unknown serostatus in regions with very highseroprevalence rates (>80%) due to serotype 1 and/or serotype 2.

The safety of the method is further described in more detail in thesection “method of preventing, method of inoculating”.

According to one embodiment such a method does not include the activesurveillance with respect to febrile illness of the subject after theadministration of the first- and second-unit dose. During activesurveillance any subject with febrile illness (defined as fever 38° C.on any 2 of 3 consecutive days) will be asked to return to the site fordengue fever evaluation by the Investigator. Subjects/guardians will becontacted at least weekly to ensure robust identification of febrileillness by reminding subjects/guardians of their obligation to return tothe site in case of febrile illness. This contact will be implementedthrough appropriate methods that may differ in each trial site (eg,phone calls, text messaging, home visits, school-based surveillance).

According to one embodiment such a method does not include vaccineimmunogenicity analysis including GMTs for dengue neutralizingantibodies.

According to one embodiment such a method does not include areactogenicity analysis. Such a reactogenicity analysis relates tosolicited local AEs (injection site pain, injection site erythema, andinjection site swelling) and solicited systemic AEs (child <6 years:fever, irritability/fussiness, drowsiness and loss of appetite; child 6years: asthenia, fever, headache, malaise and myalgia) which will e.g.be assessed for 7 days and 14 days, respectively, following eachvaccination (vaccination day included) via collection of diary cards.

According to one embodiment the method does not include an activesurveillance, an immunogenicity analysis and a reactogenicity analysis.

According to such embodiments the vaccine and the corresponding methodof inoculation are safe and therefore do not require further steps ofsurveillance or analysis.

In view of the above the method according to one embodiment comprises aprimary vaccination consisting of the steps of:

(A) selecting a subject for administration of the unit doses of thetetravalent dengue virus composition in need for protection againstdengue infection without determination of a previous dengue infection,and(B) administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and(C) administering a second unit dose of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.Therefore the method of inoculating is finalized without determinationof a previous dengue infection. The method further optionally comprisesat least 1 years after the administration of the second unit dose abooster dose of the unit dose.Selecting the subject may include all types of considerations butpreferably not the determination of a previous dengue infection. Theselection may include consideration of the age, health conditions, andthreat of infection. The threat of infection includes consideration ofthe seroprevalence rate in the region in which the subject normallylives or intends to travel, the serotype specific seroprevalence rateand an outbreak situation or serotype specific outbreak situations. Thesubject may be selected due to its exposure to serotype 1 and/orserotype 2 or due to the fact it requires protection against a specificdengue serotype, i.e. serotype 1 and/or serotype 2.

According to the invention the method is applicable to subjects of allkinds of ages. According to one embodiment the subject is under 9 yearsof age, or 4 to 5 years of age, or 6 to 11 years of age or 12 to 16years, or 6 to 16 years of age or 4 to 16 years of age, or 2 to 17 yearsof age, or 9 years of age, or over 9 years of age, or 9 to 17 years ofage, or 18 to 45 years of age, or 46 to 60 years of age, or over 60years of age.

In particular the present invention is directed to such a method whereinthe method which is safe.

In particular the present invention is directed to such a methodproviding a combined vaccine efficacy against virologically-confirmeddengue with hospitalization against all four serotypes with a 2-sided95% confidence interval, wherein the lower bound is more than 65%, whenmeasured against placebo in a subject population of at least 5,000healthy 4 to 16 year old subjects irrespective of serostatus at baselinefrom first administration of the administration schedule until 12 to 18months after the last administration of the administration schedule.

In particular the present invention is directed to such a method whereinthe method which is effective.

In particular the present invention is directed to such a methodproviding a combined vaccine efficacy against all four serotypes, inpreventing virologically confirmable dengue disease with a 2-sided 95%confidence interval, wherein the lower bound is more than 60%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects irrespective of serostatus at baseline and 14 to 16years of age, from the first administration of the administrationschedule until 18 months after the last administration of theadministration schedule.

In certain embodiments, the invention is directed to said methods,wherein the subject is seronegative to all dengue serotypes.

The present invention is directed in part to a method of preventingdengue disease (in particular virologically confirmable dengue, VCD) ina subject population. Thus, in certain embodiments the invention isdirected to a method of preventing dengue disease in a subjectpopulation, comprising administering to the subject population a unitdose, in particular a reconstituted unit dose of the invention asdescribed herein.

The present invention is in part directed to said method for preventingdengue disease (in particular virologically confirmable dengue, VCD) ina subject population comprising administering to the subject populationat least a first reconstituted unit dose of the invention as describedherein, wherein certain ratios of geometric mean neutralizing antibodytiters (GMTs) at day 180 or 365 after administration of said first unitdose to the subject population are achieved. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease (in particular virologically confirmable dengue, VCD) ina subject comprising administering to the subject at least a firstreconstituted unit dose of the invention as described herein, whereincertain ratios of neutralizing antibody titers at day 180 or 365 afteradministration of said first unit dose to the subject are achieved.According to some embodiments, the neutralizing antibody titer fordengue serotype 2 and the neutralizing antibody titer for dengueserotype 4 at day 180 or day 365 after at least a first administrationof the reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of neutralizing antibody titer for DENV-2:neutralizingantibody titer for GMT DENV-4 of not more than 50, or not more than 40,or not more than 30, or not more than 20. In some of these embodiments,the ratio of the neutralizing antibody titers of DENV-2:DENV-1 is notmore than 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

The geometric mean neutralizing antibody titers (GMTs) of a subjectpopulation or the neutralizing antibody titers of a subject aredetermined in accordance with the microneutralization test disclosedherein, for example according to the method described in Example 2.Without wishing to be bound to any theory, it is presently understoodthat a method inducing a more balanced immune response due to theadministration of the reconstituted unit dose of the invention asdescribed herein, in terms of less differences between the geometricmean neutralizing antibody titers (GMTs) against the four dengueserotypes or the neutralizing antibody titers against the four dengueserotypes, is beneficial to the subject or subject population to bevaccinated. In particular, it is understood that a much greater responseto any one of the four serotypes, such as to DENV-2 in comparison to theother serotypes, is less beneficial.

The present invention is in part directed to said method for preventingdengue disease (in particular virologically confirmable dengue, VCD) ina subject or subject population wherein the method provides aseropositivity rate in a subject population of at least 50 subjectsincluding the administration of two unit doses subcutaneously at day 1and at day 90, wherein the subjects of the subject population areseronegative to all dengue serotypes at baseline. In certain suchembodiments, at least 80% of the subject population are seropositive forall four dengue serotypes at least one month after administration of thefirst unit dose, such as at day 30, and/or at least 80% of the subjectpopulation are seropositive for all four dengue serotypes before or atthe time of the administration of the second unit dose, such as at day90, and/or at least 80%, or at least 85%, or at least 90%, or at least95% of the subject population are seropositive for all four dengueserotypes after the administration of the second unit dose, such as atday 120, and/or at least 80%, or at least 85%, or at least 90% of thesubject population are seropositive for all four dengue serotypes afterthe administration of the second unit dose, such as at day 270.

The present invention is in part directed to said method for preventingdengue disease (in particular virologically confirmable dengue, VCD) ina subject or subject population wherein the method provides aseropositivity rate in a subject population of at least 100 subjectsincluding administration of two unit doses subcutaneously at day 1 andat day 90, wherein the subjects of the subject population comprises from20% to 40% subjects who are seronegative to all dengue serotypes andfrom 60% to 80% subjects who are seropositive to at least one dengueserotype at base line, wherein at day 120 and/or day 270 theseropositivity rate for all four dengue serotypes in the seronegativepart of the subject population and the seropositivity rate for all fourdengue serotypes in the seropositive part of the subject population donot deviate more than 10%-points and/or wherein at day 120 theseropositivity rate for all four dengue serotypes in the seronegativepart of the subject population and the seropositivity rate for all fourdengue serotypes in the seropositive part of the subject population donot deviate more than 5%-points.

The present invention is in part directed to a method of preventingvirologically confirmable dengue disease in a subject or subjectpopulation comprising administering to the subject or subject populationa reconstituted unit dose of a tetravalent dengue virus compositionincluding four live, attenuated dengue serotypes, in particular thevirus strains as described herein.

The present invention is in part directed to a method of preventingvirologically confirmable dengue disease with hospitalization in asubject or subject population comprising administering to the subject orsubject population a reconstituted unit dose of a tetravalent denguevirus composition including four live, attenuated dengue serotypes, inparticular the virus strains as described herein.

In certain embodiments, the method includes a reconstituted unitdose/tetravalent dengue virus composition of a dengue vaccinecomposition administered for preventing dengue disease in a subject or asubject population, the reconstituted unit dose comprising: atetravalent virus composition including four live attenuated denguevirus strains, wherein a unit dose is lyophilized and uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluent thereconstituted unit dose is obtained which comprises:

(i) a dengue serotype 1, such as a chimeric dengue serotype 2/1 strain,in a concentration of at least 3.3 log 10 pfu/0.5 ml,(ii) a dengue serotype 2, such as a dengue serotype 2 strain, in aconcentration of at least 2.7 log 10 pfu/0.5 ml,(iii) a dengue serotype 3, such as a chimeric dengue serotype 2/3strain, in a concentration of at least 4.0 log 10 pfu/0.5 ml, and(vi) a dengue serotype 4, such as a chimeric dengue serotype 2/4 strain,in a concentration of at least 4.5 log 10 pfu/0.5 ml.

It is preferred that the reconstituted unit dose/tetravalent denguevirus composition is used in the method of preventing dengue disease ofthe present invention, wherein upon reconstitution of the unit dose witha pharmaceutically acceptable diluent (i), (ii), (iii), and (iv) providea total concentration of pfu/0.5 mL and based on said totalconcentration the concentration of (ii) in pfu/0.5 mL is less than 2%,the concentration of (iv) in pfu/0.5 mL is at least 50%, theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 6% and wherein the subject or subjectpopulation is of 18 to 60 years of age.

In another preferred embodiment, the reconstituted unit dose/tetravalentdengue virus composition is used in the method of preventing denguedisease of the present invention, wherein upon reconstitution with apharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide atotal concentration of pfu/0.5 mL and based on said total concentrationthe concentration of (ii) in pfu/0.5 mL is less than 10%, and theconcentration of (iv) in pfu/0.5 mL is at least 50%, and theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 8% and wherein the subject or subjectpopulation is of 2 to 17 years of age.

In certain embodiments, the invention is directed to said methods,wherein said unit dose comprises a tetravalent dengue virus compositionincluding four live attenuated dengue serotypes, in particular the virusstrains described herein wherein the serotypes have certainconcentrations as described herein with respect to the virus compositionand unit dose such as:

-   -   (i) a dengue serotype 1 (e.g. chimeric dengue serotype 2/1        strain) has a concentration of 3.3 log 10 pfu/dose to 5.0 log 10        pfu/dose, or 3.3 log 10 pfu/0.5 mL to 5.0 log 10 pfu/0.5 mL    -   (ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a        concentration of 2.7 log 10 pfu/dose to 4.9 log 10 pfu/0.5 dose,        or 2.7 log 10 pfu/0.5 ml to 4.9 log 10 pfu/0.5 ml    -   (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2/3        strain) has a concentration of 4.0 log 10 pfu/dose to 5.7 log 10        pfu/0.5 dose, or 4.0 log 10 pfu/0.5 mL to 5.7 log 10 pfu/0.5 mL        and    -   (iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2/4        strain) has a concentration of 4.5 log 10 pfu/dose to 5.5 log 10        pfu/0.5 dose, or 4.5 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL.        In preferred such embodiments, the subject or subject population        is of 2 to 17 years of age, such as 4 to 16 years of age, and        preferably less than 9 years of age. In other preferred        embodiments, the subject or subject population is 4-5 years of        age, 6-11 years of age or 12-16 years of age.

In certain embodiments, the invention is directed to said methods,wherein said unit dose upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent has a concentration of 3.3 log 10pfu/0.5 mL to 3.6 log 10 pfu/0.5 mL for dengue serotype 1 (e.g. chimericdengue serotype 2/1 strain), has a concentration of 2.7 log 10 pfu/0.5mL to 4.0 log 10 pfu/0.5 mL for dengue serotype 2 (e.g. dengue serotype2 strain), has a concentration of 4.0 log 10 pfu/0.5 mL to 4.6 log 10pfu/0.5 mL for dengue serotype 3 (e.g. chimeric dengue serotype 2/3strain) and has a concentration of 4.5 log 10 pfu/0.5 mL or 4.6 log 10pfu/0.5 mL to 5.1 log 10 pfu/0.5 mL for dengue serotype 4 (e.g. chimericdengue serotype 2/4 strain). In preferred such embodiments, the subjector subject population is of 2 to 17 years of age, such as 4 to 16 yearsof age, and preferably less than 9 years of age. In other preferredembodiments, the subject or subject population is 4-5 years of age, 6-11years of age or 12-16 years of age.

In certain embodiments, the invention is directed to said methods,wherein the concentration of the dengue serotype 1 (e.g. chimeric dengueserotype 2/1 strain) measured in pfu/0.5 mL is 1% to 7% of the totalconcentration, the concentration of the dengue serotype 2 (e.g. dengueserotype 2 strain) measured in pfu/0.5 mL is less than 8% of the totalconcentration, such as in the range of 1% to 8% of the totalconcentration, the concentration of the dengue serotype 3 (e.g. chimericdengue serotype 2/3 strain) measured in pfu/0.5 mL is at least 10% ofthe total concentration, and the concentration of the dengue serotype 4(e.g. chimeric dengue serotype 2/4 strain) measured in pfu/0.5 mL is atleast 65% of the total concentration, such as in the range of 65% to80%. In certain such embodiments, the arithmetic sum of all fourserotypes is in the range of 4.6 log 10 pfu/0.5 mL to 6.7 log 10 pfu/0.5mL, preferably in the range of 4.6 log 10 pfu/0.5 mL to 5.5 log 10pfu/0.5 mL Preferably, in said embodiments the subject or subjectpopulation is of 2 to 17 years of age, such as 4 to 16 years of age, andeven more preferably less than 9 years of age. In other preferredembodiments, the subject or subject population is 4-5 years of age, 6-11years of age or 12-16 years of age.

In a further preferred embodiment, the invention is directed to saidmethods, wherein the dengue serotype 1 (e.g. chimeric dengue serotype2/1 strain) such as TDV-1 and the dengue serotype 2 (e.g. dengueserotype 2 strain) such as TDV-2 are present each in a concentrationbased on the total concentration in pfu/0.5 mL which is within 5%-pointsof each other and/or are together less than about 10% of the totalconcentration in pfu/0.5 mL. In certain such embodiments the dengueserotype 3 (e.g. chimeric dengue serotype 2/3 strain) such as TDV-3 ispreferably at least about 10% of the total concentration in pfu/0.5 mLand more preferably the dengue serotype 4 (e.g. chimeric dengue serotype2/4 strain) such as TDV-4 is at least about 70% of the totalconcentration in pfu/0.5 mL. In certain such embodiments the dengueserotype 4 (e.g. chimeric dengue serotype 2/4 strain) such as TDV-4represents the highest concentration in the composition of all fourserotypes, preferably with at least about 70% of the total concentrationin pfu/0.5 mL, dengue serotype 3 (e.g. chimeric dengue serotype 2/3strain) such as TDV-3 represents the second highest concentration in thecomposition of all four serotypes, preferably with at least about 10% ofthe total concentration in pfu/0.5 mL, and dengue serotype 1 (e.g.chimeric dengue serotype 2/1 strain) such as TDV-1 and dengue serotype 2(e.g. dengue serotype 2 strain) such as TDV-2 each represent lowerconcentrations than the concentration of serotype 3 (e.g. chimericdengue serotype 2/3 strain) such as TDV-3, and optionally togetherrepresent less than about 10% of the total concentration in pfu/0.5 mL.

Preferably, the chimeric dengue serotype 2/1 strain is TDV-1, the dengueserotype 2 strain is TDV-2, the chimeric dengue serotype 2/3 strain isTDV-3 and the chimeric dengue serotype 2/4 strain is TDV-4. Morepreferably, TDV-1 is characterized by the nucleotide sequence accordingto SEQ ID No. 1 and the amino acid sequence according to SEQ ID No. 2,TDV-2 is characterized by the nucleotide sequence according to SEQ IDNo. 3 and the amino acid sequence according to SEQ ID No. 4, TDV-3 ischaracterized by the nucleotide sequence according to SEQ ID No. 5 andthe amino acid sequence according to SEQ ID No. 6 and TDV-4 ischaracterized by the nucleotide sequence according to SEQ ID No. 7 andthe amino acid sequence according to SEQ ID No. 8.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered by subcutaneous injection. According to some of theseembodiments, the subcutaneous injection is administered to the arm,preferably to the deltoid region of the arm.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose is administered to a subject ofunknown serostatus and/or wherein no test has been carried out todetermine whether the subject is seropositive or seronegative (before)the unit dose as described herein is administered. In certainembodiments, the invention is directed to said methods which do notinclude a step of determination of a previous dengue infection in thesubject or subjects. In certain embodiments, the invention is directedto said methods which do not include the analysis of the seroprevalencerate in the region or is conducted in a region with a seroprevalence ofbelow 80%, below 70% or below 60%. In certain embodiments the inventionis directed to a method wherein the serostatus of the subject isunknown. In such embodiments the serostatus is not determined at anytime before and after administration in relation to this method. Incertain embodiments of the invention the method is used in an outbreaksituation. In certain embodiments, the invention is directed to saidmethods being conducted outside a clinical trial

In certain embodiments, the invention is directed to said methods,wherein the subject, or subject population is seronegative to all dengueserotypes.

In certain embodiments, the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments the two unit doses are administeredwithin 12 months or more, or within six months, or within three months,and optionally at least 4 weeks apart such as at day 0 and day 90 or atday 1 and day 90. According to some of these embodiments, a furtherthird unit dose of the invention as described herein is administeredafter the second administration. Such a third administration may act asa booster and may be administered between 6 to 12 months after the firstadministration, such as 12 months after the first administration, orlater than 12 month after the first administration, such as 12 months (1year) after the second administration or even 5 years or longer afterthe first or second administration.

In certain embodiments, the method of the invention comprises orconsists of a single unit dose of the invention being administered.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously to a subject or subject population thatis seronegative with respect to all dengue serotypes. In otherembodiments, the subject or subject population is seropositive withrespect to at least one dengue serotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein isadministered to a subject or subject population from a dengue endemicregion. In some of these embodiments, the subject or subject populationis from Singapore, Dominican Republic, Panama, Philippines, Colombia,Puerto Rico or Thailand, in particular from Singapore, DominicanRepublic, Panama, or Philippines. In a preferred embodiment, the subjector subject population is from Asia Pacific or from Latin America. Insome other of these embodiments, the subject or subject population isfrom Thailand, Sri Lanka, Philippines, Panama, Nicaragua, DominicanRepublic, Colombia or Brazil. In other embodiments, the subject, orsubject population is from a dengue non-endemic region. Such a subjectpopulation or such a subject may be vaccinated according to the presentinvention in the context of traveling to a dengue endemic region. Incertain embodiments, the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject, or subjectpopulation that is from a dengue endemic region or a dengue non-endemicregion.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously to a subject or subject population of 2to 60 years of age. In some embodiments, the subjects or subjectpopulation are adults of more than 17 years, or more than 18 years, or18 to 60 years. In further specific embodiments, the subjects or subjectpopulation are adults of more than 21 years, or 21 to 60 years, or 21 to45 years of age.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously to children and adolescents of 2 to 17years of age. In some embodiments, the subjects or subject populationare less than 9 years of age, or less than 4 years of age. In someembodiments, the subjects or subject population are from 2 to 9 years ofage, or from 2 to 5 years of age, or from 4 to 9 years of age or from 6to 9 years of age. In other embodiment, the subject or subjectpopulation is 4 to 16 years of age. In some such embodiments, thesubject or subject population is 4-5 years of age, 6-11 years of age or12-16 years of age. Optionally, the subject or subject population isseronegative with respect to all dengue serotypes.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein isadministered to a pediatric subject or pediatric subject population ofless than 2 years of age, preferably of 2 months to 2 years or 2 monthsto 1.5 years or 2 months to 1 year. According to some of theseembodiments, the pediatric subject or pediatric subject population isseronegative and from a dengue endemic region.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered to a pediatric subject or pediatric subject populationof less than 2 years of age, preferably of 2 months to 2 years or 2months to 1.5 years or 2 months to 1 year, preferably by subcutaneousinjection. According to some of these embodiments, the pediatric subjector pediatric subject population is seronegative and from a dengueendemic region.

In a certain embodiments, the invention is directed to said methods,wherein the subject or subject population is 4-5 years of age and fromAsia Pacific, 6-11 years of age and from Asia Pacific, or 12-16 years ofage and from Asia Pacific. In other embodiments, the subject or subjectpopulation is 4-5 years of age and from Latin America, 6-11 years of ageand from Latin America, or 12-16 years of age and from Latin America.

In a certain embodiments, the invention is directed to said methods,wherein the subject or subject population is 4-5 years of age andseropositive for at least 1 dengue serotype, 6-11 years of age andseropositive for at least 1 dengue serotype, or 12-16 years of age andseropositive for at least 1 dengue serotype. In other embodiments, thesubject or subject population is 4-5 years of age and seronegative forall dengue serotypes, 6-11 years of age and seronegative for all dengueserotypes, or 12-16 years of age and seronegative for all dengueserotypes.

In a certain embodiments, the invention is directed to said methods,wherein the subject or subject population is from Asia Pacific or LatinAmerica and seropositive for at least one dengue serotype at baseline.In other embodiments, the subject or subject population is from AsiaPacific or Latin America and seronegative for at all dengue serotype atbaseline.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is from Asia Pacific,seropositive for at least one dengue serotype at baseline and 4-5 yearsof age, 6-11 years of age, or 12-16 years of age. In other embodiments,the subject or subject population is from Asia Pacific, seronegative forall dengue serotypes at baseline and 4-5 years of age, 6-11 years ofage, or 12-16 years of age. In yet other embodiments, the subject orsubject population is from Latin America, seropositive for at least onedengue serotype at baseline and 4-5 years of age, 6-11 years of age, or12-16 years of age. In other embodiments, the subject or subjectpopulation is from America, seronegative for all dengue serotypes atbaseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population had prior vaccination againstYellow Fever. In other embodiments, the subject or subject populationhad prior vaccination against Japanese Encephalitis. In yet otherembodiments, the subject or subject population had no prior vaccinationagainst Yellow Fever. In other embodiments, the subject or subjectpopulation had no prior vaccination against Japanese Encephalitis. Priorvaccination indicates a vaccination prior to 30 days after a secondadministration, such as within 4 months after the first administration,with the reconstituted unit dose as described herein. For example forvaccine efficacy (VE) as determined in Example 6 from 30 dayspost-second vaccination, a prior vaccination of Yellow Fever is definedas a Yellow Fever vaccination occurring before 30 days post-secondvaccination. In certain embodiments, the subject or subject populationreceived Dengvaxia® within the administration regimen as describedherein or within 4.5 years after administration of the first dose.

Particularly unbalanced titers of neutralizing antibodies against thefour dengue serotypes are observed in seronegative populations orsubjects after administration of the commercially available denguevaccine. The present invention shows that in particular seronegativesubjects show a more balanced immune response to the four dengueserotypes after administration of the reconstituted unit dose of theinvention as described herein. It is therefore contemplated that theunit dose of the invention as described herein and methods of thepresent invention as described herein may provide a more robust immuneresponse in a subject population including both seropositive andseronegative subjects. This balanced response and balanced efficacy andsafety is required to allow inoculation without prior serostatusanalysis which is a major advantage in vaccination programs and inparticular in outbreak situations.

The present invention is directed in part to a method of preventingvirologically confirmable dengue disease in a subject comprisingadministering to the subject a tetravalent dengue virus compositionincluding four dengue virus strains representing serotype 1, serotype 2,serotype 3 and serotype 4, wherein the virus strains are optionallylive, attenuated dengue virus strains.

The present invention is directed in part to a method of preventingvirologically confirmable dengue disease in a subject consisting ofadministering to the subject a tetravalent dengue virus compositionincluding four dengue virus strains representing serotype 1, serotype 2,serotype 3 and serotype 4, wherein the virus strains are optionallylive, attenuated dengue virus strains.

In certain embodiments, the invention is directed to said methods,wherein there is no step of determining the serostatus of the subject atbaseline, in other words, said methods do not comprise a determinationof a previous dengue infection of the subject at baseline before theadministration of the tetravalent dengue virus composition. Inparticular, such methods are safe and effective. Thus, in certain suchembodiments, the subject has not been tested for the presence a previousdengue infection.

In certain embodiments, the invention is directed to said methods,wherein the vaccine administration is safe irrespective of whether thereis a determination that the subject had a previous dengue infectionbefore the administration of the tetravalent dengue virus composition.In particular, such methods are also effective.

In certain embodiments, the invention is directed to said methods,wherein the method is safe and/or effective.

In certain embodiments, the invention is directed to said methods,wherein the composition includes at least one chimeric dengue virus. Incertain such embodiments, the invention is directed to said methods,wherein the composition includes at least one non-chimeric dengue virusand at least one chimeric dengue virus, in particular a chimeric dengueserotype 2/1 strain and a dengue serotype 2 strain and a chimeric dengueserotype 2/3 strain and a chimeric dengue serotype 2/4 strain. Thedetails of the composition are described above.

Therefore, in certain embodiments, the invention is directed to saidmethods having a vaccine efficacy, preferably a combined vaccineefficacy against all four serotypes, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 5,000 healthy subjects (or at least10,000, or at least 15,000 healthy subjects) irrespective of serostatusat baseline and e.g. 14 to 16 years of age, wherein a reconstituted unitdose/tetravalent dengue virus composition as described herein or placebois administered e.g. at least twice within less than 6 months, such aswithin 3 months, after first administration or 30 days after thesecond/last administration until at least 12 to 18 months (e.g. at 12 orat 18 months) after the second/last administration. In embodiments, theinvention is directed to said methods having a vaccine efficacy,preferably a combined vaccine efficacy against all four serotypes, inpreventing virologically confirmable dengue disease with a 2-sided 95%confidence interval, wherein the lower bound is more than 25%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline, wherein areconstituted unit dose or tetravalent dengue virus composition asdescribed herein or placebo is administered at least once, until 15 to21 months (e.g. 15 or 21 months) after the first administration of theadministration schedule. In certain such embodiments, the lower bound ismore than 30%, more than 40%, more than 50%, more than 55%, more than60%, more than 65%, more than 70% or more than 72%. Preferably saidreconstituted unit dose or placebo is administered subcutaneously withinabout 3 months, such as on days 0 and 90.

Therefore, in certain embodiments, the invention is directed to saidmethods having a combined vaccine efficacy against all four serotypes,in preventing virologically confirmable dengue disease with a 2-sided95% confidence interval, wherein the lower bound is more than 60%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is administered e.g. at leasttwice within less than 6 months, such as within 3 months, after thefirst administration until 18 months after the last administration. Inthese embodiments, the lower bound is e.g. more than 62%, more than 64%,more than 66%, more than 68%, or more than 69%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy, preferably a combined vaccine efficacy against allfour serotypes, in preventing virologically confirmable dengue diseaseof more than 30%, when measured against placebo in a subject populationof at least 5,000 healthy subjects (or at least 10,000, or at least15,000 healthy subjects) irrespective of serostatus at baseline and e.g.14 to 16 years of age, wherein a reconstituted unit dose or tetravalentdengue virus composition as described herein or placebo is administeredat least twice within less than 6 months, such as within 3 months, afterfirst administration or 30 days after the second administration/lastadministration until at least 12 months or until 12 to 18 months (e.g.at 12 or at 18 months) after the second administration/lastadministration. In certain embodiments, the invention is directed tosaid methods having a vaccine efficacy, preferably a combined vaccineefficacy against all four serotypes, in preventing virologicallyconfirmable dengue disease of more than 30%, when measured againstplacebo in a subject population of at least 5,000 healthy subjects (orat least 10,000, or at least 15,000 healthy subjects) irrespective ofserostatus at baseline, wherein a reconstituted unit dose or tetravalentdengue virus composition as described herein or placebo is administeredat least once, until 15 months after the first administration of theadministration schedule. In certain such embodiments, the vaccineefficacy is more than 40%, more than 50%, more than 55%, more than 60%,more than 65%, more than 70%, more than 75%, more than 78%, more than79% or about 80%. Preferably said reconstituted unit dose or placebo isadministered subcutaneously within about 3 month, such as on days 0 and90.

Therefore, in certain embodiments, the invention is directed to saidmethods having a combined vaccine efficacy against all four serotypes,in preventing virologically confirmable dengue disease of more than 66%,when measured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 14 to 16 years ofage, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is administered e.g. at leasttwice within less than 6 months, such as within 3 months, after thefirst administration until 18 months after the last administration. Inthese embodiments, the vaccine efficacy is e.g. more than 68%, more than70%, more than 72%, or more than 74%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy, preferably a combined vaccine efficacy against allfour serotypes, in preventing virologically confirmable dengue diseasewith hospitalization with a 2-sided 95% confidence interval, wherein thelower bound is more than 0%, when measured against placebo in a subjectpopulation of at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) irrespective of serostatus at baseline,wherein a reconstituted unit dose or tetravalent dengue viruscomposition as described herein or placebo is administered at leasttwice within less than 6 months, such as within 3 months, 30 days afterthe second administration until at least 18 months after the secondadministration. In certain such embodiments, the lower bound is morethan 10%, is more than 20%, is more than 30%, is more than 40%, is morethan 50%, is more than 55%, is more than 60%, is more than 65%, is morethan 70% or is more than 80%, or more than 90%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes inseronegative subjects with a 2-sided 95% confidence interval, whereinthe lower bound is more than 25%, when measured against placebo in asubject population of at least 1,500 or at least 2,000 healthy subjectsbeing seronegative against all serotypes at baseline, wherein said unitdose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 50%, or is morethan 55%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 1,500 or at least2,000 or at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) being seronegative against all serotypesat baseline and 4 to 16 years of age, wherein a reconstituted unitdose/tetravalent dengue virus composition as described herein or placebois e.g. administered at least twice within less than 6 months, such aswithin 3 months, from 30 days post last administration until 12 to 18months (e.g. at 12 months or at 18 months) after the lastadministration. In certain such embodiments, the lower bound is morethan 30%, is more than 35%, is more than 40%, or is more than 45%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 5,000 healthysubjects (or at least 10,000, or at least 15,000 healthy subjects) beingseropositive at baseline and 4 to 16 years of age, wherein areconstituted unit dose/tetravalent dengue virus composition asdescribed herein or placebo is e.g. administered at least twice withinless than 6 months, such as within 3 months, from 30 days post lastadministration until 12 to 18 months (e.g. at 12 months or at 18 months)after the last administration. In certain such embodiments, the lowerbound is more than 40%, is more than 45%, is more than 50%, is more than60%, or is more than 65%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 5,000 healthysubjects (or at least 10,000, or at least 15,000 healthy subjects) beingseropositive at baseline being or seronegative against all serotypes atbaseline and 4 to 16 years of age, wherein a reconstituted unitdose/tetravalent dengue virus composition as described herein or placebois e.g. administered at least twice within less than 6 months, such aswithin 3 months, from 30 days post last administration until 12 to 18months (e.g. at 12 months or at 18 months) after the lastadministration. In certain such embodiments, the difference between thelower bound provided by the seropositive subjects at baseline and thesubjects seronegative against all serotypes at baseline is no more than15%-points.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes inseronegative subjects of more than 30%, when measured against placebo ina subject population of at least 1,500 or at least 2,000 healthysubjects being seronegative against all serotypes at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsor until 12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the combined vaccineefficacy against all four dengue serotypes in seronegative subjects ismore than 40%, is more than 50%, is more than 60%, is more than 65%, oris more than 70%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease, when measured against placeboin a subject population of at least 1,500 or at least 2,000 or at least5,000 healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seronegative against all serotypes at baseline and 4 to16 years of age, wherein a reconstituted unit dose/tetravalent denguevirus composition as described herein or placebo is e.g. administered atleast twice within less than 6 months, such as within 3 months, from 30days post last administration until 12 to 18 months (e.g. at 12 monthsor at 18 months) after the last administration. In certain suchembodiments the said vaccine efficacy is more than 30%, is more than40%, is more than 50%, is more than 55%, is more than 60%, or is morethan 65%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease, when measured against placeboin a subject population of at least 5,000 healthy subjects (or at least10,000, or at least 15,000 healthy subjects) being seropositive atbaseline and 4 to 16 years of age, wherein a reconstituted unitdose/tetravalent dengue virus composition as described herein or placebois e.g. administered at least twice within less than 6 months, such aswithin 3 months, from 30 days post last administration until 12 to 18months (e.g. at 12 months or at 18 months) after the lastadministration. In certain such embodiments the said vaccine efficacy ismore than 40%, is more than 50%, is more than 60%, is more than 65%, ismore than 70%, or is more than 75%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 5,000 healthysubjects (or at least 10,000, or at least 15,000 healthy subjects) beingseropositive at baseline being or seronegative against all serotypes atbaseline and 4 to 16 years of age, wherein a reconstituted unitdose/tetravalent dengue virus composition as described herein or placebois e.g. administered at least twice within less than 6 months, such aswithin 3 months, from 30 days post last administration until 12 to 18months (e.g. at 12 months or at 18 months) after the lastadministration. In certain such embodiments, the difference between thelower bound provided by the seropositive subjects at baseline and thesubjects seronegative against all serotypes at baseline is no more than15%-points, or is no more than 10%-points.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least1,000 healthy subjects 4 to 5 years of age at the time of randomizationand irrespective of serostatus at baseline, wherein said unitdose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 45%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes of morethan 30%, when measured against placebo in a subject population of atleast 1,000 healthy subjects 4 to 5 years of age at the time ofrandomization and irrespective of serostatus at baseline, wherein saidunit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsor until 12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the combined vaccineefficacy against all four dengue serotypes is more than 40%, is morethan 50%, is more than 60%, is more than 65%, or is more than 70%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least1,000 healthy subjects 6 to 11 years of age at the time of randomizationand irrespective of serostatus at baseline, wherein said unitdose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 50%, is more than60%, or is more than 70%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes of morethan 30%, when measured against placebo in a subject population of atleast 1,000 healthy subjects 6 to 11 years of age at the time ofrandomization and irrespective of serostatus at baseline, wherein saidunit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsor until 12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the combined vaccineefficacy against all four dengue serotypes is more than 40%, is morethan 50%, is more than 60%, is more than 70%, is more than 75%, or ismore than 80%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least1,000 healthy subjects 12 to 16 years of age at the time ofrandomization and irrespective of serostatus at baseline, wherein saidunit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 50%, is more than60%, is more than 65%, or is more than 68%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four dengue serotypes of morethan 30%, when measured against placebo in a subject population of atleast 1,000 healthy subjects 12 to 16 years of age at the time ofrandomization and irrespective of serostatus at baseline, wherein saidunit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsor until 12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the combined vaccineefficacy against all four dengue serotypes is more than 40%, is morethan 50%, is more than 60%, is more than 70%, is more than 75%, or ismore than 80%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against dengue serotype 1 with a 2-sided 95%confidence interval, wherein the lower bound is more than 25%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects, or at least 10,000 healthy subjects, or at least15,000 healthy subjects irrespective of serostatus at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, or is more than 50%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 1, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is e.g. administered at leasttwice within less than 6 months, such as within 3 months, from 30 dayspost last administration until 12 to 18 months (e.g. at 12 or at 18months) after the last administration. In certain such embodiments, thelower bound is more than 30%, is more than 35% is more than 40%, is morethan 45%, is more than 50%, or is more than 54%. In certain suchembodiments the subject population of at least 1,500 is seronegativeagainst all serotypes at base line and the lower bound is more than 35%.In certain such embodiments the seronegative and seropositive populationeach provide a vaccine efficacy against serotype 1 with a 2-sided 95%confidence interval, wherein the lower bounds are within 10%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against dengue serotype 1 of more than 30%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects, or at least 10,000 healthy subjects, or at least15,000 healthy subjects irrespective of serostatus at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsor until 12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the vaccine efficacyagainst dengue serotype 1 is more than 40%, is more than 50%, is morethan 60%, is more than 65%, or is more than 70%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 1, in preventing virologicallyconfirmable dengue disease, when measured against placebo in a subjectpopulation of at least 1,500, or at least 2,000, or at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is e.g. administered at leasttwice within less than 6 months, such as within 3 months, from 30 dayspost last administration until 12 to 18 months (e.g. at 12 or at 18months) after the last administration. In certain such embodiments, thevaccine efficacy is more than 40%, is more than 50%, is more than 60%,or is more than 65%. In certain such embodiments the subject populationof at least 1,500 is seronegative against all serotypes at base line. Incertain such embodiments the seronegative and seropositive populationeach provide a vaccine efficacy against serotype 1 which are within5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against dengue serotype 2 with a 2-sided 95%confidence interval, wherein the lower bound is more than 25%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects, or at least 10,000 healthy subjects, or at least15,000 healthy subjects irrespective of serostatus at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 50, is more than60, is more than 70, is more than 80, or is more than 90%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 2, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is e.g. administered at leasttwice within less than 6 months, such as within 3 months, from 30 dayspost last administration until 12 to 18 months (e.g. at 12 or at 18months) after the last administration. In certain such embodiments, thelower bound is more than 50%, is more than 60%, is more than 70%, ismore than 80%, or is more than 85%. In certain such embodiments thesubject population of at least 1,500, is seronegative against allserotypes. In certain such embodiments the seronegative and seropositivepopulation each provide a vaccine efficacy against serotype 2 with a2-sided 95% confidence interval, wherein the lower bounds are within5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against dengue serotype 2 of more than 30%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects, or at least 10,000 healthy subjects, or at least15,000 healthy subjects irrespective of serostatus at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsor until 12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the vaccine efficacyagainst dengue serotype 2 is more than 40%, is more than 50%, is morethan 60%, is more than 70%, is more than 80, or is more than 90%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 2, in preventing virologicallyconfirmable dengue disease, when measured against placebo in a subjectpopulation of at least 1,500, or at least 2,000, or at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is e.g. administered at leasttwice within less than 6 months, such as within 3 months, from 30 dayspost last administration until 12 to 18 months (e.g. at 12 or at 18months) after the last administration. In certain such embodiments, thevaccine efficacy is more than 60%, is more than 70%, is more than 80%,or is more than 90%. In certain such embodiments the subject populationof at least 1,500 is seronegative against all serotypes at base line. Incertain such embodiments the seronegative and seropositive populationeach provide a vaccine efficacy against serotype 2 which are within5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against dengue serotype 3 with a 2-sided 95%confidence interval, wherein the lower bound is more than 25%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects, or at least 10,000 healthy subjects, or at least15,000 healthy subjects irrespective of serostatus at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months after the secondadministration of the administration schedule. In certain suchembodiments, the lower bound is more than 30%, is more than 40%.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against dengue serotype 3 of more than 30%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects, or at least 10,000 healthy subjects, or at least15,000 healthy subjects irrespective of serostatus at baseline, whereinsaid unit dose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, 30 days after the second administration until at least 12 monthsafter the second administration. In certain such embodiments, thevaccine efficacy against dengue serotype 3 is more than 40%, is morethan 50%, is more than 55%, or is more than 60%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least5,000 healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is e.g. administered at leasttwice within less than 6 months, such as within 3 months, from firstadministration until 12 to 18 months (e.g. at 12 months or at 18 months)after the last administration, or from 30 days post last administrationuntil 12 to 18 months (e.g. at 12 or at 18 months) after the lastadministration. In certain such embodiments, the lower bound is morethan 10%, is more than 20%, is more than 30%, is more than 40%, is morethan 50%, is more than 55%, is more than 60%, is more than 65%, is morethan 66%, is more than 67%, is more than 70%, is more than 75%, is morethan 77%, or is more than 80%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization, whenmeasured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage, wherein a reconstituted unit dose/tetravalent dengue viruscomposition as described herein or placebo is e.g. administered at leasttwice within less than 6 months, such as within 3 months, from firstadministration until 12 to 18 months (e.g. at 12 months or at 18 months)after the last administration, or from 30 days post last administrationuntil 12 to 18 months (e.g. at 12 or at 18 months) after the lastadministration. In certain such embodiments, the vaccine efficacy ismore than is more than 70%, is more than 75%, is more than 80%, or ismore than 82%, or is more than 85%, more than 88%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 1,500 or at least2,000 healthy subjects being seronegative against all serotypes atbaseline, wherein said unit dose/tetravalent dengue virus composition orsaid placebo is administered at least twice within less than 6 months,such as within 3 months, about 30 days after the second administrationof the administration schedule until at least 12 months or until 12 to18 months (e.g. at 12 or at 18 months) after the second administrationof the administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 50%, is more than60%, is more than 70%, or is more than 75%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least5,000 healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seronegative against all serotypes at baseline and 4 to16 years of age, wherein a reconstituted unit dose/tetravalent denguevirus composition as described herein or placebo is e.g. administered atleast twice within less than 6 months, such as within 3 months, from 30days post last administration until 12 to 18 months (e.g. at 12 monthsor at 18 months) after the last administration. In certain suchembodiments, the lower bound is more than 60%, is more than 65%, is morethan 66%, is more than 67%, is more than 70%, is more than 75%, is morethan 77% or is more than 80%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes of more than 30%, whenmeasured against placebo in a subject population of at least 1,500 or atleast 2,000 healthy subjects, healthy subjects being seronegativeagainst all serotypes at baseline, wherein said unit dose/tetravalentdengue virus composition or said placebo is administered at least twicewithin less than 6 months, such as within 3 months, 30 days after thesecond administration until at least 12 months or until 12 to 18 months(e.g. at 12 or at 18 months) after the second administration. In certainsuch embodiments, the combined vaccine efficacy againstvirologically-confirmed dengue with hospitalization against all fourserotypes is more than 40%, is more than 50%, is more than 60%, is morethan 70%, is more than 80%, or is more than 90%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization, whenmeasured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seronegative against all serotypes at baseline and 4 to16 years of age, wherein a reconstituted unit dose/tetravalent denguevirus composition as described herein or placebo is e.g. administered atleast twice within less than 6 months, such as within 3 months, from 30days post last administration until 12 to 18 months (e.g. at 12 monthsor at 18 months) after the last administration. In certain suchembodiments, the said vaccine efficacy is more than 60%, is more than65%, is more than 66%, is more than 67%, is more than 70%, is more than75%, is more than 77%, is more than 80, or is more than 85%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 1,500 or at least2,000 healthy subjects being seropositive at baseline, wherein said unitdose/tetravalent dengue virus composition or said placebo isadministered at least twice within less than 6 months, such as within 3months, about 30 days after the second administration of theadministration schedule until at least 12 months or until 12 to 18months (e.g. at 12 or at 18 months) after the second administration ofthe administration schedule. In certain such embodiments, the lowerbound is more than 30%, is more than 40%, is more than 50%, is more than60%, is more than 70%, or is more than 80%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least5,000 healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seropositive at baseline and 4 to 16 years of age,wherein a reconstituted unit dose/tetravalent dengue virus compositionas described herein or placebo is e.g. administered at least twicewithin less than 6 months, such as within 3 months, from 30 days postlast administration until 12 to 18 months (e.g. at 12 months or at 18months) after the last administration. In certain such embodiments, thelower bound is more than 60%, is more than 65%, is more than 70%, ismore than 75%, or is more than 80%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes of more than 30%, whenmeasured against placebo in a subject population at least 1,500 or of atleast 2,000 healthy subjects, healthy subjects being seropositive atbaseline, wherein said unit dose/tetravalent dengue virus composition orsaid placebo is administered at least twice within less than 6 months,such as within 3 months, 30 days after the second administration untilat least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months)after the second administration. In certain such embodiments, thecombined vaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes is more than 40%, is morethan 50%, is more than 60%, is more than 70%, is more than 80%, or ismore than 90%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization, whenmeasured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seropositive at baseline and 4 to 16 years of age,wherein a reconstituted unit dose/tetravalent dengue virus compositionas described herein or placebo is e.g. administered at least twicewithin less than 6 months, such as within 3 months, from 30 days postlast administration until 12 to 18 months (e.g. at 12 months or at 18months) after the last administration. In certain such embodiments, thevaccine efficacy is more than 75%, is more than 70%, is more than 80%,is more than 85%, or is more than 90%.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization with a2-sided 95% confidence interval, wherein the lower bound is more than25%, when measured against placebo in a subject population of at least5,000 healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seropositive at baseline being or seronegative againstall serotypes at baseline and 4 to 16 years of age, wherein areconstituted unit dose/tetravalent dengue virus composition asdescribed herein or placebo is e.g. administered at least twice withinless than 6 months, such as within 3 months, from 30 days post lastadministration until 12 to 18 months (e.g. at 12 months or at 18 months)after the last administration. In certain such embodiments, thedifference between the lower bound provided by the seropositive subjectsat baseline and the subjects seronegative against all serotypes atbaseline is no more than 15%-points.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with hospitalization, whenmeasured against placebo in a subject population of at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) being seropositive at baseline being or seronegative againstall serotypes at baseline and 4 to 16 years of age, wherein areconstituted unit dose/tetravalent dengue virus composition asdescribed herein or placebo is e.g. administered at least twice withinless than 6 months, such as within 3 months, from 30 days post lastadministration until 12 to 18 months (e.g. at 12 months or at 18 months)after the last administration. In certain such embodiments, thedifference between the vaccine efficacy provided by the seropositivesubjects at baseline and the subjects seronegative against all serotypesat baseline is no more than 10%-points or no more than 5%-points.

In certain embodiments, the invention is directed to said methods havinga relative risk, preferably a combined relative risk against all fourserotypes, with a 2-sided 95% confidence interval, wherein the upperbound is less than 0.75, when measured against placebo in a subjectpopulation of at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) irrespective of serostatus at baseline,wherein a reconstituted unit dose/tetravalent dengue virus compositionas described herein or placebo is administered at least twice withinless than 6 months, such as within 3 months, 30 days after the secondadministration until at least 12 months after the second administration.In certain such embodiments, the upper bound is less than 0.70, lessthan 0.65, less than 0.60, less than 0.55, less than 0.50, less than0.45, less than 0.40, less than 0.35, less than 0.30 or less than 0.28.Preferably said reconstituted unit dose or placebo is administeredsubcutaneously within about 3 month, such as on days 0 and 90.

In certain embodiments, the invention is directed to said methods havinga relative risk, preferably a combined relative risk against all fourserotypes, of less than 0.70, when measured against placebo in a subjectpopulation of at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) irrespective of serostatus at baseline,wherein a reconstituted unit dose/tetravalent dengue virus compositionas described herein or placebo is administered at least twice withinless than 6 months, such as within 3 months, 30 days after the secondadministration until at least 12 months after the second administration.In certain such embodiments, the relative risk is less than 0.65, lessthan 0.60, less than 0.55, less than 0.50, less than 0.45, less than0.40, less than 0.35, less than 0.30, less than 0.25 or less than 0.23.Preferably said reconstituted unit dose or placebo is administeredsubcutaneously within about 3 month, such as on days 0 and 90.

In certain embodiments, the invention is directed to said methods,wherein virologically confirmable dengue disease occurs in less than2.5% of the subjects, when measured against placebo in a subjectpopulation of at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) irrespective of serostatus at baseline,wherein a reconstituted unit dose/tetravalent dengue virus compositionas described herein or placebo is administered at least twice withinless than 6 months, such as within 3 months, 30 days after the secondadministration until at least 12 months or at least 18 months after thesecond administration. In certain such embodiments, virologicallyconfirmable dengue disease occurs in less than 2.0% of the subjects,less than 1.5% of the subjects, less than 1.0% of the subjects, lessthan 0.8% of the subjects, or less than 0.6% of the subjects. Preferablysaid reconstituted unit dose or placebo is administered subcutaneouslywithin about 3 month, such as on days 0 and 90.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes with a 2-sided95% confidence interval, wherein the lower bound is more than 61.0%, ormore than 65.0 or more than 70.0% or more than 72.0% when measuredagainst placebo in a subject population of at least 5,000 healthysubjects (or at least 10,000, or at least 15,000 healthy subjects) fromendemic irrespective of serostatus at baseline and being selected fromthe group consisting of 4 to 16 year old subjects at the time ofrandomization, wherein said unit dose/tetravalent dengue viruscomposition or said placebo is administered at least twice within 6months or less, about 30 days after the last administration of theadministration schedule until at least 12 or 13 months after the lastadministration of the administration schedule.

In certain embodiments, the invention is directed to said methods havinga combined vaccine efficacy against all four serotypes of more than 66%,or of more than 70%, or of more than 75%, or of more than 77%, or ofmore than 80.0%, when measured against placebo in a subject populationof at least 5,000 healthy subjects (or at least 10,000, or at least15,000 healthy subjects) from endemic areas irrespective of serostatusat baseline and being selected from the group consisting of 4 to 16 yearold subjects at the time of randomization, wherein said unitdose/tetravalent dengue virus composition or said placebo isadministered at least twice within 6 months or less, about 30 days afterthe last administration of the administration schedule until at least 12months or 13 month after the last administration of the administrationschedule.

In certain embodiments, the invention is directed to said methods,wherein the combined vaccine efficacy against all four serotypes ismeasured about 30 days after the last administration of theadministration schedule until 12 or 13 months after the lastadministration of the administration schedule.

In certain embodiments, the invention is directed to said methods,wherein said unit dose or said placebo is administered at least twicewithin three months, in particular at about day 1 and about day 90, andwherein the combined vaccine efficacy against all four serotypes ismeasured 30 days after the second administration until 12 or 13 monthsafter the second administration of the administration schedule.

In certain embodiments, the invention is directed to said methods,wherein said methods are effective and safe. In some of theseembodiments, the subject or subject population is under 9 years of age,under 4 years of age, or under 2 years of age or from 2 to 9 years ofage, or from 2 to 5 years of age, or from 4 to 9 years of age or from 6to 9 years of age. Optionally the subject is seronegative with respectto all dengue serotypes.

In certain embodiments, the invention is directed to said methods,wherein said methods having a relative risk for virologically confirmeddengue with hospitalization of 1 or less, or 0.8 or less, or 0.6 orless, when measured against placebo in a subject population of at least5,000 healthy subjects (or at least 10,000, or at least 15,000 healthysubjects). In some of these embodiments, the subject or subjectpopulation is under 9 years of age, under 4 years of age, or under 2years of age or from 2 to 9 years of age, or from 2 to 5 years of age,or from 4 to 9 years of age or from 6 to 9 years of age. Optionally thesubject is seronegative with respect to all dengue serotypes.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are 4 to 16 yearsof age. In some of such embodiments, the healthy subjects of the subjectpopulation are 4 to 5 years of age, 6 to 11 years of age, or 12 to 16years of age.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are defined asbeing healthy in view of the exclusion criteria specified in Example 6.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are from AsiaPacific or Latin America.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are seropositivewith respect to at least one serotype. In other embodiments, the healthysubjects of the subject population are seronegative with respect to allserotypes.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are 4-5 years ofage and from Asia Pacific, 6-11 years of age and from Asia Pacific, or12-16 years of age and from Asia Pacific. In other embodiments, thehealthy subjects of the subject population are 4-5 years of age and fromLatin America, 6-11 years of age and from Latin America, or 12-16 yearsof age and from Latin America.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are 4-5 years ofage and seropositive for at least 1 dengue serotype, 6-11 years of ageand seropositive for at least 1 dengue serotype, or 12-16 years of ageand seropositive for at least 1 dengue serotype. In other embodiments,the healthy subjects of the subject population are 4-5 years of age andseronegative for all dengue serotypes, 6-11 years of age andseronegative for all dengue serotypes, or 12-16 years of age andseronegative for all dengue serotypes.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are from AsiaPacific or Latin America and seropositive for at least one dengueserotype at baseline. In other embodiments, the healthy subjects of thesubject population are from Asia Pacific or Latin America andseronegative for at all dengue serotype at baseline.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population are from AsiaPacific, seropositive for at least one dengue serotype at baseline and4-5 years of age, 6-11 years of age, or 12-16 years of age. In otherembodiments, the healthy subjects of the subject population are fromAsia Pacific, seronegative for all dengue serotypes at baseline and 4-5years of age, 6-11 years of age, or 12-16 years of age. In yet otherembodiments, the healthy subjects of the subject population are fromLatin America, seropositive for at least one dengue serotype at baselineand 4-5 years of age, 6-11 years of age, or 12-16 years of age. In otherembodiments, the healthy subjects of the subject population are fromAmerica, seronegative for all dengue serotypes at baseline and 4-5 yearsof age, 6-11 years of age, or 12-16 years of age.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population had priorvaccination against Yellow Fever. In other embodiments, the healthysubjects of the subject population had no prior vaccination againstYellow Fever. Prior vaccination indicates a vaccination prior to thefirst vaccination with the reconstituted unit dose as described herein.For example for vaccine efficacy (VE) as determined in Example 6 from 30days post-second vaccination, a prior vaccination of Yellow Fever isdefined as a Yellow Fever vaccination occurring before 30 dayspost-second vaccination.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population had priorvaccination against Japanese Encephalitis. In other embodiments, thehealthy subjects of the subject population had no prior vaccinationagainst Japanese Encephalitis.

In certain embodiments, the invention is directed to said methods,wherein the healthy subjects of the subject population receivedDengvaxia® within the administration regimen as described herein orwithin 4.5 years after administration of the first dose. In certainembodiments, the invention is directed to said methods, wherein theoccurrence of vaccine related serious adverse events is less than 0.1%.

In certain embodiments, the invention is directed to said methods,wherein the occurrence of vaccine related unsolicited adverse eventsoccurring within 4 weeks of administration is less than 2%.

In certain embodiments, the invention is directed to said methods,wherein the occurrence of vaccine related solicited adverse eventsoccurring within 2 weeks of administration is less than 35%.

In certain embodiments, the invention is directed to said methods,wherein the occurrence of vaccine related solicited local reactionsoccurring within 1 weeks of administration is less than 40%.

In certain embodiments, the invention is directed to said methods,wherein the method does not increase the risk of virologically-confirmeddengue with hospitalization in the individual, such as in a seronegativeindividual.

In one embodiment, the unit dose is co-administered with a vaccineselected from the group of a

DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine, anMMR vaccine, a yellow fever vaccine, in particular YF-17D, an HPVvaccine, in particular a 9vHPV vaccine, a tetanus, diphtheria, andpertussis (Tdap) vaccine, in particular a combined tetanus toxoid,reduced diphtheria toxoid and acellular pertussis (adsorbed) vaccine,and a hepatitis A vaccine as described in more detail below.

Unit Dose for Use in a Method of Preventing Dengue Disease

The present invention is directed in part to the composition or unitdose of the invention as described herein for use in a method ofpreventing dengue disease (in particular virologically-confirmabledengue, VCD) in a subject.

The present invention is directed in part to the composition or unitdose of the invention as described herein for use in a method ofpreventing dengue disease (in particular virologically-confirmabledengue, VCD) in a subject population.

Inoculating against dengue disease, vaccinating against dengue diseaseand preventing dengue disease have the same meaning. The presentinvention is directed in part to a method of preventing denguehemorrhagic fever (DHF) and dengue shock syndrome (DSS).

Any method described herein above under the heading “Method ofpreventing, method of inoculating” is to be understood to be alsodisclosed as unit dose for use in such a method of preventing denguedisease in a subject or subject population irrespective of beingexpressly stated below.

According to a certain aspect the composition or unit dose of theinvention is a tetravalent dengue virus composition including a chimericdengue serotype 2/1 strain and a dengue serotype 2 strain and a chimericdengue serotype 2/3 strain and a chimeric dengue serotype 2/4 strain.According to certain such aspects the dengue serotype 2 strain isderived from the wild type virus strain DEN-2 16681 (represented by SEQID NO 11) and differs in at least three nucleotides from the wild typeas follows:

a) 5′-noncoding region (NCR)-57 (nt-57 C-to-T)b) NS1-53 Gly-to-Asp (nt-2579 G-to-A)c) NS3-250 Glu-to-Val (nt-5270 A-to-T); andwherein the three chimeric dengue strains are derived from the serotype2 strain by replacing the structural proteins prM and E from serotype 2strain with the corresponding structural proteins from the other dengueserotypes, resulting in the following chimeric dengue strains:a DENV-2/1 chimera,a DENV-2/3 chimera anda DENV-2/4 chimera.

Further information regarding the serotypes of the tetravalentcomposition can be derived from section “Dengue virus strains” above.

The tetravalent dengue virus composition for such use may be in the formof a unit dose comprising:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The tetravalent dengue virus composition for such use may also be in theform of a lyophilized unit dose which upon reconstitution with 0.5 mL ofa pharmaceutically acceptable diluent comprises:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

In the tetravalent dengue virus composition for such use according toone embodiment upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is preferably less than 10%, and the concentration of(iv) in pfu/0.5 mL is preferably at least 50%, and the concentration of(i) in pfu/0.5 mL is preferably at least 1%, and the concentration of(iii) in pfu/0.5 mL is preferably at least 8%, or more preferred atleast 10%, or at least 12%, or at least 14%, or at least 16%, or atleast 18% and wherein the subject is preferably 2 to 17 years of age or4 to 16 years of age.

In the tetravalent dengue virus composition for such use according toone embodiment upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is preferably less than 2%, the concentration of (iv)in pfu/0.5 mL is preferably at least 50%, the concentration of (i) inpfu/0.5 mL is at preferably least 1%, and the concentration of (iii) inpfu/0.5 mL is preferably at least 6% and wherein the subject preferablyis 18 to 60 years of age.

Further information regarding the tetravalent composition or the unitdose can be derived from section “Dengue vaccine composition” and “Unitdose” above.

According to one embodiment the unit dose for such use is directed to amethod comprising a primary vaccination with only two administrations ofthe unit dose comprising the steps of:

(A) administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and(B) administering a second unit does of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.According to this embodiment the administration of only two doses within3 months is sufficient to provide effective protection against asubsequent dengue infection.

Such method preferably provides a combined vaccine efficacy against allfour serotypes in preventing virologically confirmable dengue diseasewith a 2-sided 95% confidence interval, wherein the lower bound is morethan 60%, when measured against placebo in a subject population of atleast 5,000 healthy subjects irrespective of serostatus at baseline and14 to 16 years of age, from the first administration of theadministration schedule until 18 months after the second administrationof the administration schedule.

Such method also preferably provides a combined vaccine efficacy againstall four serotypes, in preventing virologically confirmable denguedisease with a 2-sided 95% confidence interval, wherein the lower boundis more than 45%, when measured against placebo in a subject populationof at least 1,500 or at least 2,000 healthy subjects seronegativeagainst all serotypes at baseline and 14 to 16 years of age, from 30days after the second administration of the administration scheduleuntil 18 months after the second administration of the administrationschedule.

According to certain embodiments the use is for a method of inoculationagainst the virologically confirmable dengue disease is due to a dengueserotype 2, and/or due to a dengue serotype 1. The method has very highefficacy against dengue serotype 2 and dengue serotype 1 and the highestefficacy against dengue serotype 2.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 1, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age from 30 days post second administration until 12 to 18months (e.g. at 12 or at 18 months) after the second administration. Incertain such embodiments, the lower bound is more than 30%, is more than35% is more than 40%, is more than 45%, is more than 50%, or is morethan 54%. In certain such embodiments the subject population of at least1,500 is seronegative against all serotypes at base line and the lowerbound is more than 35%. In certain such embodiments the seronegative andseropositive population each provide a vaccine efficacy against serotype1 with a 2-sided 95% confidence interval, wherein the lower bounds arewithin 10%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 1, in preventing virologicallyconfirmable dengue disease, when measured against placebo in a subjectpopulation of at least 1,500, or at least 2,000, or at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage from 30 days post second administration until 12 to 18 months (e.g.at 12 or at 18 months) after the second administration. In certain suchembodiments, the vaccine efficacy is more than 40%, is more than 50%, ismore than 60%, or is more than 65%. In certain such embodiments thesubject population of at least 1,500 is seronegative against allserotypes at base line. In certain such embodiments the seronegative andseropositive population each provide a vaccine efficacy against serotype1 which are within 5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 2, in preventing virologicallyconfirmable dengue disease with a 2-sided 95% confidence interval,wherein the lower bound is more than 25%, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age from 30 days post second administration until 12 to 18months (e.g. at 12 or at 18 months) after the second administration. Incertain such embodiments, the lower bound is more than 50%, is more than60%, is more than 70%, is more than 80%, or is more than 85%. In certainsuch embodiments the subject population of at least 1,500, isseronegative against all serotypes. In certain such embodiments theseronegative and seropositive population each provide a vaccine efficacyagainst serotype 2 with a 2-sided 95% confidence interval, wherein thelower bounds are within 5%-points.

In certain embodiments, the invention is directed to said methods havinga vaccine efficacy against serotype 2, in preventing virologicallyconfirmable dengue disease, when measured against placebo in a subjectpopulation of at least 1,500, or at least 2,000, or at least 5,000healthy subjects (or at least 10,000, or at least 15,000 healthysubjects) irrespective of serostatus at baseline and 4 to 16 years ofage from 30 days post second administration until 12 to 18 months (e.g.at 12 or at 18 months) after the second administration. In certain suchembodiments, the vaccine efficacy is more than 60%, is more than 70%, ismore than 80%, or is more than 90%. In certain such embodiments thesubject population of at least 1,500 is seronegative against allserotypes at base line. In certain such embodiments the seronegative andseropositive population each provide a vaccine efficacy against serotype2 which are within 5%-points.

The efficacy of the method is further described in more detail in thesection “method of preventing, method of inoculating”.

In certain embodiments the unit dose is reconstituted and administeredby subcutaneous injection. According to some of these embodiments, thesubcutaneous injection is administered to the arm, preferably to thedeltoid region of the arm.

According to one embodiment such a method does not include a step ofdetermination of a previous dengue infection in the subjects preferablyat any time before, during or after the steps of administration orwherein the serostatus of the subject is unknown preferably at any timebefore, during or after the steps of administration.

The method according to the invention does not require the testing ofthe serostatus before vaccination and thus allows immediate treatmentand outbreak control. According to certain embodiments the use is for amethod wherein the subject is exposed to a dengue outbreak. In certainsuch embodiments the outbreak is due to a dengue serotype 2, and/or dueto a serotype 1.

According to one embodiment such a method the subject is from a regionwherein the seroprevalence rate is unknown and/or wherein theseroprevalence rate is below 80%, or below 70%, or below 60%.

According to one embodiment of such a method the subject is seronegativeat baseline and is from a region or travels to a region wherein theseroprevalence rate is high with respect to serotype 1 and/or serotype 2i.e. 80%, or 90% or above.

According this embodiment the vaccine and corresponding method is safefor seronegative and seropositive subjects and thus does not require ananalysis of the serostatus or a determination of a previous dengueinfection or a high seroprevalence rate in the region. Such a methodpreferably provides a combined vaccine efficacy againstvirologically-confirmed dengue with hospitalization against all fourserotypes with a 2-sided 95% confidence interval, wherein the lowerbound is more than 65%, when measured against placebo in a subjectpopulation of at least 5,000 healthy 4 to 16 year old subjectsirrespective of serostatus at baseline, preferably in at least 1,500healthy 4 to 16 year old subjects seronegative at baseline, from firstadministration of the administration schedule until 12 to 18 monthsafter the second administration of the administration schedule.Preferably, the 2-sided 95% confidence interval of the combined vaccineefficacy against virologically-confirmed dengue with hospitalizationagainst all four serotypes when comparing seropositive and seronegativesubjects provides for lower bounds of the 2-sided confidence intervalwhich are within 10% points or within 15% points or within 20% points.The method is preferably safe with respect to serotype 1 and serotype 2which may therefore be used in outbreak situations due to serotype 1and/or serotype 2 or even for seronegative subjects (e.g. travelers) orsubjects with unknown serostatus in regions with very highseroprevalence rates (>80%) due to serotype 1 and/or serotype 2.

The safety of the method is further described in more detail in thesection “method of preventing, method of inoculating”.

According to one embodiment such a method does not include the activesurveillance with respect to febrile illness of the subject after theadministration of the first- and second-unit dose. During activesurveillance any subject with febrile illness (defined as fever 38° C.on any 2 of 3 consecutive days) will be asked to return to the site fordengue fever evaluation by the Investigator. Subjects/guardians will becontacted at least weekly to ensure robust identification of febrileillness by reminding subjects/guardians of their obligation to return tothe site in case of febrile illness. This contact will be implementedthrough appropriate methods that may differ in each trial site (eg,phone calls, text messaging, home visits, school-based surveillance).

According to one embodiment such a method does not include vaccineimmunogenicity analysis including GMTs for dengue neutralizingantibodies.

According to one embodiment such a method does not include areactogenicity analysis. Such a reactogenicity analysis relates tosolicited local AEs (injection site pain, injection site erythema, andinjection site swelling) and solicited systemic AEs (child <6 years:fever, irritability/fussiness, drowsiness and loss of appetite; child 6years: asthenia, fever, headache, malaise and myalgia) which will e.g.be assessed for 7 days and 14 days, respectively, following eachvaccination (vaccination day included) via collection of diary cards.

According to one embodiment the method does not include an activesurveillance, an immunogenicity analysis and a reactogenicity analysis.

According to such embodiments the vaccine and the corresponding methodof inoculation are safe and therefore do not require further steps ofsurveillance or analysis.

In view of the above the method according to one embodiment comprises aprimary vaccination consisting of the steps of:

(A) selecting a subject for administration of the unit doses of thetetravalent dengue virus composition in need for protection againstdengue infection without determination of a previous dengue infection,and(B) administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and(C) administering a second unit dose of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.Therefore the method of inoculating is finalized without determinationof a previous dengue infection. The method however further optionallycomprises at least 1 years after the administration of the second unitdose a booster dose of the unit dose.

Selecting the subject may include all types of considerations butpreferably not the determination of a previous dengue infection. Theselection may include consideration of the age, health conditions, andthreat of infection. The threat of infection includes consideration ofthe seroprevalence rate in the region in which the subject normallylives or intends to travel, the serotype specific seroprevalence rateand an outbreak situation or serotype specific outbreak situations. Thesubject may be selected due to its exposure to serotype 1 and/orserotype 2 or due to the fact it requires protection against a specificdengue serotype, i.e. serotype 1 and/or serotype 2.

According to the invention the method is applicable to subjects of allkinds of ages. According to one embodiment the subject is under 9 yearsof age, or 4 to 5 years of age, or 6 to 11 years of age or 12 to 16years, or 6 to 16 years of age or 4 to 16 years of age, or 2 to 17 yearsof age, or 9 years of age, or over 9 years of age, or 9 to 17 years ofage, or 18 to 45 years of age, or 46 to 60 years of age, or over 60years of age.

According to certain such embodiments the unit dose of the tetravalentdengue virus composition comprising (i), (ii), (iii), and (iv) uponreconstitution with a pharmaceutically acceptable diluent provides atotal concentration of pfu/0.5 mL of (i), (ii), (iii), and (iv) andbased on said total concentration the concentration of (ii) in pfu/0.5mL is preferably less than 10%, and the concentration of (iv) in pfu/0.5mL is preferably at least 50%, and the concentration of (i) in pfu/0.5mL is preferably at least 1%, and the concentration of (iii) in pfu/0.5mL is preferably at least 8%, or more preferred at least 10%, or atleast 12%, or at least 14%, or at least 16%, or at least 18% and thesubject is 2 to 17 years of age or 4 to 16 years of age.

According to certain such embodiments the unit dose of the tetravalentdengue virus composition comprising (i), (ii), (iii), and (iv) uponreconstitution with a pharmaceutically acceptable diluent provides atotal concentration of pfu/0.5 mL of (i), (ii), (iii), and (iv) andbased on said total concentration the concentration of (ii) in pfu/0.5mL is preferably less than 2%, the concentration of (iv) in pfu/0.5 mLis preferably at least 50%, the concentration of (i) in pfu/0.5 mL is atpreferably least 1%, and the concentration of (iii) in pfu/0.5 mL ispreferably at least 6% and the subject is 18 to 60 years of age.

Further specific embodiments are described below.

The present invention is directed in part to a reconstituted unit doseof a dengue vaccine composition as described herein for use in a methodof preventing virologically confirmable dengue disease in a subjectcomprising administering at least a first unit dose of the denguevaccine composition to the subject, wherein the dengue vaccinecomposition is a tetravalent dengue virus composition including fourdengue virus strains representing dengue serotype 1, dengue serotype 2,dengue serotype 3 and dengue serotype 4, optionally wherein the denguevirus strains are live, attenuated dengue virus strains and/or comprisechimeric dengue viruses and/or at least one non-chimeric dengue virus,and wherein upon reconstitution with 0.5 mL of a pharmaceuticallyacceptable diluent

-   -   (i) dengue serotype 1 has a concentration of at least 3.3 log 10        pfu/0.5 mL and optionally to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of at least 2.7 log        10 pfu/0.5 mL and optionally to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of at least 4.0 log        10 pfu/0.5 mL and optionally to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of at least 4.5 log        10 pfu/0.5 mL and optionally to 6.2 log 10 pfu/0.5 mL.

The present invention is directed in part to a reconstituted unit doseof a dengue vaccine composition as described herein for use in a methodof preventing virologically confirmable dengue disease in a subjectcomprising consecutively administering at least a first and a secondunit dose of the dengue vaccine composition to the subject, wherein saidfirst and second unit dose are administered subcutaneously within 3months and at least 4 weeks apart, optionally at about day 1 and atabout day 90, wherein the dengue vaccine composition is a tetravalentdengue virus composition including four dengue virus strainsrepresenting dengue serotype 1, dengue serotype 2, dengue serotype 3 anddengue serotype 4, optionally wherein the dengue virus strains are live,attenuated, and wherein upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent

-   -   (v) dengue serotype 1 has a concentration of at least 3.3 log 10        pfu/0.5 mL and optionally to 5.0 log 10 pfu/0.5 mL,    -   (vi) dengue serotype 2 has a concentration of at least 2.7 log        10 pfu/0.5 mL and optionally to 4.9 log 10 pfu/0.5 mL,    -   (vii) dengue serotype 3 has a concentration of at least 4.0 log        10 pfu/0.5 mL and optionally to 5.7 log 10 pfu/0.5 mL, and    -   (viii) dengue serotype 4 has a concentration of at least 4.5 log        10 pfu/0.5 mL and optionally to 6.2 log 10 pfu/0.5 mL.

In certain embodiments, the invention is directed to a reconstitutedunit dose of a dengue vaccine composition for use in a method ofpreventing virologically confirmable dengue disease in a subjectcomprising consecutively administering at least a first and a secondunit dose of the dengue vaccine composition to the subject, wherein saidfirst and second unit dose are administered subcutaneously within 3months and at least 4 weeks apart, optionally at about day 1 and atabout day 90, wherein the dengue vaccine composition is a tetravalentdengue virus composition including four dengue virus strainsrepresenting dengue serotype 1, dengue serotype 2, dengue serotype 3 anddengue serotype 4, optionally wherein the dengue virus strains are live,attenuated, wherein the subject is under 9 years of age and/or when theserostatus of the subject is unknown or seronegative and wherein uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluent

-   -   (i) dengue serotype 1 has a concentration of at least 3.3 log 10        pfu/0.5 mL and optionally to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of at least 2.7 log        10 pfu/0.5 mL and optionally to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of at least 4.0 log        10 pfu/0.5 mL and optionally to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of at least 4.5 log        10 pfu/0.5 mL and optionally to 6.2 log 10 pfu/0.5 mL.

In certain embodiments, the reconstituted unit dose is administered to asubject of unknown serostatus and/or wherein no test has been carriedout to determine whether the subject is seropositive or seronegativebefore the unit dose as described herein is administered.

In certain embodiments, the subject is under 9 years of age and/or theserostatus of the subject is unknown or seronegative. In certain suchembodiments, the subject is under 9 years of age and the serostatus ofthe subject is unknown or seronegative, preferably seronegative.

In certain embodiments, the method is safe. In certain such embodiments,the subject is under 9 years of age or from 4 years of age and/or theserostatus of the subject is unknown or seronegative. In certain suchembodiments, the subject is from 4 years of age and the serostatus ofthe subject is unknown or seronegative, preferably seronegative.

In certain embodiments, the method is effective. In certain suchembodiments, the subject is under 9 years of age and/or the serostatusof the subject is unknown or seronegative. In certain such embodiments,the subject is under 9 years of age and the serostatus of the subject isunknown or seronegative, preferably seronegative.

In certain embodiments, the dengue serotype 1 and the dengue serotype 2are present each in a concentration based on the total concentration inpfu/0.5 mL which is within 5%-points of each other and/or are togetherless than about 10% of the total concentration in pfu/0.5 mL. In certainsuch embodiments, the dengue serotype 3 is at least about 10% of thetotal concentration in pfu/0.5 mL and/or the dengue serotype 4 is atleast about 70% of the total concentration in pfu/0.5 mL.

In certain embodiments, the dengue serotype 4 represents the highestconcentration in the composition of all four serotypes, preferably withat least about 70% of the total concentration in pfu/0.5 mL, dengueserotype 3 represents the second highest concentration in thecomposition of all four serotypes, preferably with at least about 10% ofthe total concentration in pfu/0.5 mL, and dengue serotype 1 and dengueserotype 2 each represent lower concentrations than the concentration ofserotype 3, and optionally together represent less than about 10% of thetotal concentration in pfu/0.5 mL.

In certain embodiments, the composition includes at least one chimericdengue virus. In certain such embodiments, the composition includes atleast one non-chimeric dengue virus and at least one chimeric denguevirus.

In certain embodiments, the subject is seronegative to all dengueserotypes at baseline and/or is from 4 years of age, optionally to 60years of age. In certain such embodiments, the subject is 4 to 16 yearsof age, under 9 years of age, from 2 years of age to under 9 years ofage, from 4 years of age to under 9 years of age, 4 to 5 years of age, 6to 11 years of age, or 12 to 16 years of age. In other embodiments, thesubject is seropositive to at least one dengue serotypes at baselineand/or is from 4 years of age, optionally to 60 years of age. In certainsuch embodiments, the subject is 4 to 16 years of age, under 9 years ofage, from 2 years of age to under 9 years of age, from 4 years of age tounder 9 years of age, 4 to 5 years of age, 6 to 11 years of age, or 12to 16 years of age.

In certain embodiments, the method does not comprise a determination ofa previous dengue infection in the subject before the administration ofthe first unit dose of the tetravalent dengue virus composition. Thus,in certain embodiments, the subject has not been tested for the presencea previous dengue infection.

In certain embodiments, the dengue serotype 1 is a chimeric dengueserotype 2/1 strain, the dengue serotype 2 is a non-chimeric dengueserotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype2/3 strain and the dengue serotype 4 is a chimeric dengue serotype 2/4strain and the dengue serotype 1 has the amino acid sequence of SEQ IDNO. 2, the dengue serotype 2 has the amino acid sequence of SEQ ID NO.4, the dengue serotype 3 has the amino acid sequence of SEQ ID NO. 6,and the dengue serotype 4 has the amino acid sequence of SEQ ID NO. 8.

In certain embodiments, the unit dose further comprises from about 10%w/v to about 20% w/v 0,0-trehalose dihydrate or an equimolar amount ofother forms of α,α-trehalose, from about 0.5% w/v to about 1.5% w/vpoloxamer 407, from about 0.05% w/v to about 2% w/v human serum albumin,and from about 70 mM to 140 mM sodium chloride when measured in 0.5 mL.In certain such embodiments, the unit dose comprises about 15% (w/v)α,α-trehalose dihydrate, about 1% (w/v) poloxamer 407, about 0.1% (w/v)human serum albumin, and about 100 mM sodium chloride when measured in0.5 mL.

In certain embodiments, the method is for preventing dengue hemorrhagicfever (DHF) or dengue shock syndrome (DSS).

In certain embodiments, the subject is from a dengue endemic region. Inother embodiments, the subject is from a dengue non-endemic region.

In certain embodiments, the subject is from Asia Pacific or LatinAmerica.

In certain embodiments, the reconstituted unit dose provides aseropositivity rate when it is administered to a subject population ofat least 50 subjects in two unit doses subcutaneously at day 1 and atday 90, wherein the subjects of the subject population are seronegativeto all dengue serotypes at baseline. In certain such embodiments, atleast 80% of the subject population are seropositive for all four dengueserotypes at least one month after administration of the first unitdose, such as at day 30, and/or at least 80% of the subject populationare seropositive for all four dengue serotypes before or at the time ofthe administration of the second unit dose, such as at day 90, and/or atleast 80%, or at least 85% or at least 90%, or at least 95% of thesubject population are seropositive for all four dengue serotypes afterthe administration of the second unit dose, such as at day 120, and/orat least 80%, or at least 85%, or at least 90% of the subject populationare seropositive for all four dengue serotypes after the administrationof the second unit dose, such as at day 270. In certain suchembodiments, at least 80% of the subject population are seropositive forall four dengue serotypes at least one month after administration of thefirst unit dose, such as at day 30, and before or at the time of theadministration of the second unit dose, such as at day 90, and after theadministration of the second unit dose, such as at day 120 and at day270.

In certain embodiments, the reconstituted unit dose provides aseropositivity rate, when it is administered to a subject population ofat least 100 subjects in two unit doses subcutaneously at day 1 and atday 90, wherein the subjects of the subject population comprises from20% to 40% subjects who are seronegative to all dengue serotypes andfrom 60% to 80% subjects who are seropositive to at least one dengueserotype at base line, wherein at day 120 and/or day 270 theseropositivity rate for all four dengue serotypes in the seronegativepart of the subject population and the seropositivity rate for all fourdengue serotypes in the seropositive part of the subject population donot deviate more than 10%-points and/or wherein at day 120 theseropositivity rate for all four dengue serotypes in the seronegativepart of the subject population and the seropositivity rate for all fourdengue serotypes in the seropositive part of the subject population donot deviate more than 5%-points.

In certain particular embodiments, the invention is directed to a denguevaccine composition as described herein for use in a method ofpreventing virologically confirmable dengue disease in a subjectcomprising consecutively administering at least a first and a secondunit dose of the dengue vaccine composition to the subject, wherein saidfirst and second unit dose are administered subcutaneously within 3months and at least 4 weeks apart, optionally at about day 1 and atabout day 90, and wherein the dengue vaccine composition is atetravalent dengue virus composition including four live, attenuateddengue virus strains representing dengue serotype 1, dengue serotype 2,dengue serotype 3 and dengue serotype 4, wherein the attenuated denguevirus strains comprise chimeric dengue viruses and at least onenon-chimeric dengue virus, and wherein the dengue serotype 1 and thedengue serotype 2 are present each in a concentration based on the totalconcentration in pfu/0.5 mL which is within 5%-points of each otherand/or are together less than about 10% of the total concentration inpfu/0.5 mL.

In certain embodiments, the method does not comprise a determination ofa previous dengue infection of the subject before the administration ofthe first unit dose of the tetravalent dengue virus composition andwherein the method is safe and effective. Thus, in certain embodiments,the subject has not been tested for the presence a previous dengueinfection.

In certain embodiments, the dengue serotype 3 is at least about 10% ofthe total concentration in pfu/0.5 mL and/or the dengue serotype 4 is atleast about 70% of the total concentration in pfu/0.5 mL.

In certain embodiments, the dengue serotype 4 represents the highestconcentration in the composition of all four serotypes, preferably withat least about 70% of the total concentration in pfu/0.5 mL, dengueserotype 3 represents the second highest concentration in thecomposition of all four serotypes, preferably with at least about 10% ofthe total concentration in pfu/0.5 mL, and dengue serotype 1 and dengueserotype 2 each represent lower concentrations than the concentration ofserotype 3, and optionally together represent less than about 10% of thetotal concentration in pfu/0.5 mL.

In certain embodiments, the dengue serotype 1 is a chimeric dengueserotype 2/1 strain, the dengue serotype 2 is a non-chimeric dengueserotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype2/3 strain and the dengue serotype 4 is a chimeric dengue serotype 2/4strain and the dengue serotype 1 has the amino acid sequence of SEQ IDNO. 2, the dengue serotype 2 has the amino acid sequence of SEQ ID NO.4, the dengue serotype 3 has the amino acid sequence of SEQ ID NO. 6,and the dengue serotype 4 has the amino acid sequence of SEQ ID NO. 8.

In certain embodiments, in the unit dose upon reconstitution with 0.5 mLof a pharmaceutically acceptable diluent

-   -   (i) dengue serotype 1 has a concentration of 3.3 log 10 pfu/0.5        mL to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of 2.7 log 10 pfu/0.5        mL to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of 4.0 log 10        pfu/0.5 mL to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of 4.5 log 10 pfu/0.5        mL to 6.2 log 10 pfu/0.5 mL, and optionally the composition        further comprises about 15% (w/v) α,α-trehalose dihydrate, about        1% (w/v) poloxamer 407, about 0.1% (w/v) human serum albumin,        and about 100 mM sodium chloride when measured in 0.5 mL.

In certain embodiments, the unit doses are administered to the deltoidregion of the arm.

In certain embodiments, the composition is administered withoutdetermining the serostatus of the subject at baseline and wherein theadministration is safe and effective regardless of the serostatus atbase line.

In certain embodiments, the subject is seronegative to all dengueserotypes at baseline and/or is from 4 years of age, optionally to 60years of age. In certain such embodiments, the subject is 4 to 16 yearsof age, under 9 years of age, from 2 years of age to under 9 years ofage, from 4 years of age to under 9 years of age, 4 to 5 years of age, 6to 11 years of age, or 12 to 16 years of age. In particular the subjectmay be under 9 years of age and seronegative to all four dengueserotypes at baseline. In other embodiments, the subject is seropositiveto at least one dengue serotypes at baseline and/or is from 4 years ofage, optionally to 60 years of age. In certain such embodiments, thesubject is 4 to 16 years of age, under 9 years of age, from 2 years ofage to under 9 years of age, from 4 years of age to under 9 years ofage, 4 to 5 years of age, 6 to 11 years of age, or 12 to 16 years ofage. In particular the subject may be under 9 years of age andseropositive to at least one dengue serotypes at baseline. In certainpreferred embodiments, the subject is 4 to 5 years of age, 6 to 11 yearsof age or 12 to 16 years of age.

In certain embodiments, the method is for preventing dengue hemorrhagicfever (DHF) or dengue shock syndrome (DSS).

In certain embodiments, the subject is from a dengue endemic region orfrom a dengue non-endemic region.

In certain embodiments, the subject is from Asia Pacific or LatinAmerica.

In certain embodiments, the composition provides a seropositivity ratewhen it is administered to a subject population of at least 50 subjectsin two unit doses subcutaneously at day 1 and at day 90, wherein thesubjects of the subject population are seronegative to all dengueserotypes at baseline, in particular wherein at least one month afteradministration of the first unit dose, such as at day 30, at least 80%of the subject population are seropositive for all four dengueserotypes, and/or at least 80% of the subject population areseropositive for all four dengue serotypes before or at the time of theadministration of the second unit dose, such as at day 90, and/or atleast 80%, or at least 85% or at least 90%, or at least 95% of thesubject population are seropositive for all four dengue serotypes afterthe administration of the second unit dose, such as at day 120, and/orat least 80%, or at least 85%, or at least 90% of the subject populationare seropositive for all four dengue serotypes after the administrationof the second unit dose, such as at day 270.

In certain embodiments, the composition provides a seropositivity rate,when it is administered to a subject population of at least 100 subjectsin two unit doses subcutaneously at day 1 and at day 90, wherein thesubjects of the subject population comprises from 20% to 40% subjectswho are seronegative to all dengue serotypes and from 60% to 80%subjects who are seropositive to at least one dengue serotype at baseline, wherein at day 120 and/or day 270 the seropositivity rate for allfour dengue serotypes in the seronegative part of the subject populationand the seropositivity rate for all four dengue serotypes in theseropositive part of the subject population do not deviate more than10%-points and/or wherein at day 120 the seropositivity rate for allfour dengue serotypes in the seronegative part of the subject populationand the seropositivity rate for all four dengue serotypes in theseropositive part of the subject population do not deviate more than5%-points.

The present invention is in part directed to the unit dose of theinvention as described herein for use in a method of preventing denguedisease (in particular virologically confirmable dengue, VCD) in asubject population comprising administering to the subject population atleast a first reconstituted unit dose of the invention as describedherein, wherein certain ratios of geometric mean neutralizing antibodytiters (GMTs) at day 180 or 365 after administration of said first unitdose to the subject population are achieved. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the unit dose of theinvention as described herein for use in a method of preventing denguedisease (in particular virologically confirmable dengue, VCD) in asubject comprising administering to the subject at least a firstreconstituted unit dose of the invention as described herein, whereincertain ratios of neutralizing antibody titers at day 180 or 365 afteradministration of said first unit dose to the subject are achieved.According to some embodiments, the neutralizing antibody titer fordengue serotype 2 and the neutralizing antibody titer for dengueserotype 4 at day 180 or day 365 after at least a first administrationof the reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of neutralizing antibody titer for DENV-2:neutralizingantibody titer for GMT DENV-4 of not more than 50, or not more than 40,or not more than 30, or not more than 20. In some of these embodiments,the ratio of the neutralizing antibody titers of DENV-2:DENV-1 is notmore than 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

The geometric mean neutralizing antibody titers (GMTs) of a subjectpopulation or the neutralizing antibody titers of a subject aredetermined in accordance with the microneutralization test disclosedherein, for example according to the method described in Example 2.

In certain embodiments the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinsaid unit dose is administered by subcutaneous injection. According tosome of these embodiments the subcutaneous injection is administered tothe arm, preferably to the deltoid region of the arm.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein thesubject or subject population is seronegative to all dengue serotypes.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein asingle unit dose of the invention as described herein is administered.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein tworeconstituted unit doses of the invention as described herein areadministered. In some embodiments, the two reconstituted unit doses areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0 and day 90 or at day 1 and day 90.According to some of these embodiments, a third reconstituted unit doseof the invention as described herein may be administered after thesecond administration. Such a third administration may act as a boosterand may be administered between 6 to 12 months after the firstadministration, such as 12 months after the first administration, orlater than 12 month after the first administration, such as 12 months (1year) after the second administration or even 5 years or longer afterthe first or second administration.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein thereconstituted unit dose of the invention as described herein isadministered at most in two doses or in one dose.

In certain embodiments of the invention the subject is seronegative withrespect to all dengue serotypes.

In certain embodiments of the invention the subject is seronegative withrespect to all dengue serotypes and the reconstituted unit dose isadministered to the seronegative subject by subcutaneous injection.

In certain other embodiments of the invention the subject isseropositive with respect to at least one dengue serotype.

In certain embodiments the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered to a subject or subject population from a dengue endemicregion. In some of these embodiments, the subject or subject populationis from Singapore, Dominican Republic, Panama, Philippines, Colombia,Puerto Rico or Thailand, in particular from Singapore, DominicanRepublic, Panama, or Philippines. In other embodiments, the subject orsubject population is from a dengue non-endemic region. Such a subjectpopulation or such a subject may be vaccinated according to theinvention in the context of traveling to a dengue-endemic region. Incertain embodiments, the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject or subjectpopulation from a dengue endemic region or from a dengue non-endemicregion.

In some embodiments the invention is directed to the unit dose of theinvention as described herein for said uses, wherein the subject orsubject population is of 2 to 60 years of age, or more than 17 years, ormore than 18 years, or 18 to 60 years of age. In certain embodiments,the subject or subject population is of 1 to 17 years of age, or lessthan 9 years of age, or less than 4 years of age or less than 2 years ofage. In further specific embodiments, the subjects or subject populationare adults of more than 21 years, or 21 to 60 years, or 21 to 45 yearsof age. According to some of these embodiments the subject or subjectpopulation is seronegative and from a dengue-endemic region.

In certain embodiments, the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinthe unit dose of the invention as described herein is administered to apediatric subject or pediatric subject population of less than 2 yearsof age, preferably of 2 months to 2 years of age or 2 months to 1.5years of age or 2 months to 1 year of age. According to some of theseembodiments, the pediatric subject or pediatric subject population isseronegative and from a dengue endemic region.

In certain embodiments, the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinthe reconstituted unit dose is administered subcutaneously to apediatric subject or pediatric subject population of less than 2 yearsof age, preferably of 2 months to 2 years of age or 2 months to 1.5years of age or 2 months to 1 year of age. According to some of theseembodiments, the pediatric subject or pediatric subject population isseronegative and from a dengue endemic region.

The unit dose for use in the methods described above may be any unitdose of a dengue vaccine composition as described above under theheadings “Unit dose” or “Dengue vaccine composition” and comprise anydengue virus strain as described above under the heading “Dengue virusstrain”.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguehemorrhagic fever (DHF) and dengue shock syndrome (DSS) in an elderlysubject.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein theelderly subject is seronegative to all dengue serotypes.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein theelderly subject is seronegative to all dengue serotypes.

In certain embodiments the invention is directed to a reconstituted unitdose of the invention as described herein for said uses, wherein tworeconstituted unit doses of the invention as described herein areadministered. In some embodiments, the two reconstituted unit doses areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0/1 and day 90. According to some of theseembodiments, a third reconstituted unit dose of the invention asdescribed herein may be administered after the second administration.Such a third administration may act as a booster and may be administeredbetween 6 to 12 months after the first administration, such as 12 monthsafter the first administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments of the invention the elderly subject isseronegative with respect to all dengue serotypes. In certainembodiments of the invention the elderly subject is seronegative withrespect to all dengue serotypes and the reconstituted unit dose isadministered to the seronegative subject by subcutaneous injection.

In certain other embodiments of the invention the elderly subject isseropositive with respect to at least one dengue serotype.

In certain embodiments the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered to an elderly subject from a dengue endemic region. In someof these embodiments, the elderly subject is from Singapore, DominicanRepublic, Panama, Philippines, Colombia, Puerto Rico or Thailand, inparticular from Singapore, Dominican Republic, Panama, or Philippines.In other embodiments, the elderly subject is from a dengue non-endemicregion. Such an elderly subject may be vaccinated according to theinvention in the context of traveling to a dengue-endemic region. Incertain embodiments, the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to an elderly subjectfrom a dengue endemic region or from a dengue non-endemic region.

In certain embodiments, the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinthe elderly subject has at least one chronic condition or disease. Theat least one chronic condition or disease may be selected from diabetes,hypertension, allergies, previous strokes, ischemic heart disease,chronic renal impairment and chronic obstructive pulmonary disease.

In certain embodiments, the invention is directed to the reconstitutedunit dose of the invention as described herein for said uses, whereinthe elderly subject has an impaired immune system.

Use for the Manufacture of a Medicament for Preventing Dengue Disease

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease (in particular virologically confirmabledengue, VCD) in a subject.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease (in particular virologically confirmabledengue, VCD) in a subject population.

The present invention is therefore directed to the use of a tetravalentdengue virus composition including four live attenuated dengue virusstrains representing serotype 1, serotype 2, serotype 3 and serotype 4in the manufacture of a medicament for the method of inoculating asubject against virologically confirmable dengue disease, wherein inparticular the tetravalent dengue virus composition includes a chimericdengue serotype 2/1 strain and a dengue serotype 2 strain and a chimericdengue serotype 2/3 strain and a chimeric dengue serotype 2/4 strain,wherein in particular the dengue serotype 2 strain is derived from thewild type virus strain DEN-2 16681 (SEQ ID NO 11) and differs in atleast three nucleotides from the wild type as follows:

d) 5′-noncoding region (NCR)-57 (nt-57 C-to-T): major attenuation locus

e) NS1-53 Gly-to-Asp (nt-2579 G-to-A): major attenuation locus

f) NS3-250 Glu-to-Val (nt-5270 A-to-T): major attenuation locus; and

wherein the three chimeric dengue strains are derived from the serotype2 strain by replacing the structural proteins prM and E from serotype 2strain with the corresponding structural proteins from the other dengueserotypes, resulting in the following chimeric dengue strains:

-   -   a DENV-2/1 chimera,    -   a DENV-2/3 chimera and    -   a DENV-2/4 chimera.

Further information regarding the serotypes of the tetravalentcomposition can be derived from section “Dengue virus strains” above.

The present invention is in particular directed to such use wherein thetetravalent dengue virus composition is in the form of a unit dosecomprising:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The present invention is in particular directed to such use wherein theunit dose is lyophilized and upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent comprises:

(i) a dengue serotype 1 in a concentration of at least 3.3 log 10pfu/0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10pfu/0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 log 10pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.

The present invention is also in particular directed to such use whereinupon reconstitution with a pharmaceutically acceptable diluent (i),(ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL andbased on said total concentration the concentration of (ii) in pfu/0.5mL is less than 10%, and the concentration of (iv) in pfu/0.5 mL is atleast 50%, and the concentration of (i) in pfu/0.5 mL is at least 1%,and the concentration of (iii) in pfu/0.5 mL is at least 8%, or at least10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%,and wherein the subject is preferably 2 to 17 years of age or 4 to 16years of age.

The present invention is also in particular directed to such use whereinupon reconstitution with a pharmaceutically acceptable diluent (i),(ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL andbased on said total concentration the concentration of (ii) in pfu/0.5mL is less than 2%, the concentration of (iv) in pfu/0.5 mL is at least50%, the concentration of (i) in pfu/0.5 mL is at least 1%, and theconcentration of (iii) in pfu/0.5 mL is at least 6% and wherein thesubject preferably is 18 to 60 years of age.

Further information regarding the tetravalent composition or the unitdose can be derived from section

“Dengue vaccine composition” and “Unit dose” above.

According to one embodiment the use is directed to a method comprising aprimary vaccination with only two administrations of the unit dosecomprising the steps of:

(A) administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and(B) administering a second unit does of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.According to this embodiment the administration of only two doses within3 months is sufficient to provide effective protection against asubsequent dengue infection.

Such method preferably provides a combined vaccine efficacy against allfour serotypes in preventing virologically confirmable dengue diseasewith a 2-sided 95% confidence interval, wherein the lower bound is morethan 60%, when measured against placebo in a subject population of atleast 5,000 healthy subjects irrespective of serostatus at baseline and14 to 16 years of age, from the first administration of theadministration schedule until 18 months after the second administrationof the administration schedule.

Such method also preferably provides a combined vaccine efficacy againstall four serotypes, in preventing virologically confirmable denguedisease with a 2-sided 95% confidence interval, wherein the lower boundis more than 45%, when measured against placebo in a subject populationof at least 1,500 or at least 2,000 healthy subjects seronegativeagainst all serotypes at baseline and 14 to 16 years of age, from 30days after the second administration of the administration scheduleuntil 18 months after the second administration of the administrationschedule.

According to certain embodiments the use is for a method of inoculationagainst the virologically confirmable dengue disease is due to a dengueserotype 2, and/or due to a dengue serotype 1. The method has very highefficacy against dengue serotype 2 and dengue serotype 1 and the highestefficacy against dengue serotype 2.

In certain embodiments, the invention is directed to the use whereinsaid methods have a vaccine efficacy against serotype 1, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 1,500, or at least2,000, or at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) irrespective of serostatus at baselineand 4 to 16 years of age from 30 days post second administration until12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the lower bound is morethan 30%, is more than 35% is more than 40%, is more than 45%, is morethan 50%, or is more than 54%. In certain such embodiments the subjectpopulation of at least 1,500 is seronegative against all serotypes atbase line and the lower bound is more than 35%. In certain suchembodiments the seronegative and seropositive population each provide avaccine efficacy against serotype 1 with a 2-sided 95% confidenceinterval, wherein the lower bounds are within 10%-points.

In certain embodiments, the invention is directed to said use whereinsaid methods have a vaccine efficacy against serotype 1, in preventingvirologically confirmable dengue disease, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age from 30 days post second administration until 12 to 18months (e.g. at 12 or at 18 months) after the second administration. Incertain such embodiments, the vaccine efficacy is more than 40%, is morethan 50%, is more than 60%, or is more than 65%. In certain suchembodiments the subject population of at least 1,500 is seronegativeagainst all serotypes at base line. In certain such embodiments theseronegative and seropositive population each provide a vaccine efficacyagainst serotype 1 which are within 5%-points.

In certain embodiments, the invention is directed to said use whereinsaid methods have a vaccine efficacy against serotype 2, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 25%, when measuredagainst placebo in a subject population of at least 1,500, or at least2,000, or at least 5,000 healthy subjects (or at least 10,000, or atleast 15,000 healthy subjects) irrespective of serostatus at baselineand 4 to 16 years of age from 30 days post second administration until12 to 18 months (e.g. at 12 or at 18 months) after the secondadministration. In certain such embodiments, the lower bound is morethan 50%, is more than 60%, is more than 70%, is more than 80%, or ismore than 85%. In certain such embodiments the subject population of atleast 1,500, is seronegative against all serotypes. In certain suchembodiments the seronegative and seropositive population each provide avaccine efficacy against serotype 2 with a 2-sided 95% confidenceinterval, wherein the lower bounds are within 5%-points.

In certain embodiments, the invention is directed to said use whereinsaid methods have a vaccine efficacy against serotype 2, in preventingvirologically confirmable dengue disease, when measured against placeboin a subject population of at least 1,500, or at least 2,000, or atleast 5,000 healthy subjects (or at least 10,000, or at least 15,000healthy subjects) irrespective of serostatus at baseline and 4 to 16years of age from 30 days post second administration until 12 to 18months (e.g. at 12 or at 18 months) after the second administration. Incertain such embodiments, the vaccine efficacy is more than 60%, is morethan 70%, is more than 80%, or is more than 90%. In certain suchembodiments the subject population of at least 1,500 is seronegativeagainst all serotypes at base line. In certain such embodiments theseronegative and seropositive population each provide a vaccine efficacyagainst serotype 2 which are within 5%-points.

The efficacy of the method is further described in more detail in thesection “method of preventing, method of inoculating”.

In certain embodiments the unit dose is reconstituted and administeredby subcutaneous injection. According to some of these embodiments, thesubcutaneous injection is administered to the arm, preferably to thedeltoid region of the arm.

According to one embodiment such use is directed to a method which doesnot include a step of determination of a previous dengue infection inthe subjects preferably at any time before, during or after the steps ofadministration or wherein the serostatus of the subject is unknownpreferably at any time before, during or after the steps ofadministration.

The method according to the invention does not require the testing ofthe serostatus before vaccination and thus allows immediate treatmentand outbreak control. According to certain embodiments the use is for amethod wherein the subject is exposed to a dengue outbreak. In certainsuch embodiments the outbreak is due to a dengue serotype 2, and/or dueto a serotype 1.

According to one embodiment such a method the subject is from a regionwherein the seroprevalence rate is unknown and/or wherein theseroprevalence rate is below 80%, or below 70%, or below 60%.

According to one embodiment of such a method the subject is seronegativeat baseline and is from a region or travels to a region wherein theseroprevalence rate is high with respect to serotype 1 and/or serotype 2i.e. 80%, or 90% or above.

According this embodiment the vaccine and corresponding method is safefor seronegative and seropositive subjects and thus does not require ananalysis of the serostatus or a determination of a previous dengueinfection or a high seroprevalence rate in the region. Such a methodpreferably provides a combined vaccine efficacy againstvirologically-confirmed dengue with hospitalization against all fourserotypes with a 2-sided 95% confidence interval, wherein the lowerbound is more than 65%, when measured against placebo in a subjectpopulation of at least 5,000 healthy 4 to 16 year old subjectsirrespective of serostatus at baseline, preferably in at least 1,500healthy 4 to 16 year old subjects seronegative at baseline, from firstadministration of the administration schedule until 12 to 18 monthsafter the second administration of the administration schedule.Preferably, the 2-sided 95% confidence interval of the combined vaccineefficacy against virologically-confirmed dengue with hospitalizationagainst all four serotypes when comparing seropositive and seronegativesubjects provides for lower bounds of the 2-sided confidence intervalwhich are within 10% points or within 15% points or within 20% points.The method is preferably safe with respect to serotype 1 and serotype 2which may therefore be used in outbreak situations due to serotype 1and/or serotype 2 or even for seronegative subjects (e.g. travelers) orsubjects with unknown serostatus in regions with very highseroprevalence rates (>80%) due to serotype 1 and/or serotype 2.

The safety of the method is further described in more detail in thesection “method of preventing, method of inoculating”.

According to one embodiment such a method does not include the activesurveillance with respect to febrile illness of the subject after theadministration of the first- and second-unit dose. During activesurveillance any subject with febrile illness (defined as fever 38° C.on any 2 of 3 consecutive days) will be asked to return to the site fordengue fever evaluation by the Investigator. Subjects/guardians will becontacted at least weekly to ensure robust identification of febrileillness by reminding subjects/guardians of their obligation to return tothe site in case of febrile illness. This contact will be implementedthrough appropriate methods that may differ in each trial site (eg,phone calls, text messaging, home visits, school-based surveillance).

According to one embodiment such a method does not include vaccineimmunogenicity analysis including GMTs for dengue neutralizingantibodies.

According to one embodiment such a method does not include areactogenicity analysis. Such a reactogenicity analysis relates tosolicited local AEs (injection site pain, injection site erythema, andinjection site swelling) and solicited systemic AEs (child <6 years:fever, irritability/fussiness, drowsiness and loss of appetite; child 6years: asthenia, fever, headache, malaise and myalgia) which will e.g.be assessed for 7 days and 14 days, respectively, following eachvaccination (vaccination day included) via collection of diary cards.

According to one embodiment the method does not include an activesurveillance, an immunogenicity analysis and a reactogenicity analysis.

According to such embodiments the vaccine and the corresponding methodof inoculation are safe and therefore do not require further steps ofsurveillance or analysis.

In view of the above according to one embodiment the use is directed toa method comprises a primary vaccination consisting of the steps of:

(A) selecting a subject for administration of the unit doses of thetetravalent dengue virus composition in need for protection againstdengue infection without determination of a previous dengue infection,and(B) administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and(C) administering a second unit dose of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.Therefore the method of inoculating is finalized without determinationof a previous dengue infection. The method however further optionallycomprises at least 1 years after the administration of the second unitdose a booster dose of the unit dose.

Selecting the subject may include all types of considerations butpreferably not the determination of a previous dengue infection. Theselection may include consideration of the age, health conditions, andthreat of infection. The threat of infection includes consideration ofthe seroprevalence rate in the region in which the subject normallylives or intends to travel, the serotype specific seroprevalence rateand an outbreak situation or serotype specific outbreak situations. Thesubject may be selected due to its exposure to serotype 1 and/orserotype 2 or due to the fact it requires protection against a specificdengue serotype, i.e. serotype 1 and/or serotype 2.

According to the invention the method is applicable to subjects of allkinds of ages. According to one embodiment the subject is under 9 yearsof age, or 4 to 5 years of age, or 6 to 11 years of age or 12 to 16years, or 6 to 16 years of age or 4 to 16 years of age, or 2 to 17 yearsof age, or 9 years of age, or over 9 years of age, or 9 to 17 years ofage, or 18 to 45 years of age, or 46 to 60 years of age, or over 60years of age.

According to certain such embodiments the unit dose of the tetravalentdengue virus composition comprising (i), (ii), (iii), and (iv) uponreconstitution with a pharmaceutically acceptable diluent provides atotal concentration of pfu/0.5 mL of (i), (ii), (iii), and (iv) andbased on said total concentration the concentration of (ii) in pfu/0.5mL is preferably less than 10%, and the concentration of (iv) in pfu/0.5mL is preferably at least 50%, and the concentration of (i) in pfu/0.5mL is preferably at least 1%, and the concentration of (iii) in pfu/0.5mL is preferably at least 8%, or more preferred at least 10%, or atleast 12%, or at least 14%, or at least 16%, or at least 18% and thesubject is 2 to 17 years of age or 4 to 16 years of age.

According to certain such embodiments the unit dose of the tetravalentdengue virus composition comprising (i), (ii), (iii), and (iv) uponreconstitution with a pharmaceutically acceptable diluent provides atotal concentration of pfu/0.5 mL of (i), (ii), (iii), and (iv) andbased on said total concentration the concentration of (ii) in pfu/0.5mL is preferably less than 2%, the concentration of (iv) in pfu/0.5 mLis preferably at least 50%, the concentration of (i) in pfu/0.5 mL is atpreferably least 1%, and the concentration of (iii) in pfu/0.5 mL ispreferably at least 6% and the subject is 18 to 60 years of age.

Any method described herein above under the heading “Method ofpreventing” is to be understood to be also disclosed as the use of aunit dose for the manufacture of a medicament for preventing denguedisease in a subject or subject population with such a methodirrespective of whether it is expressly stated below.

Further specific embodiments are described below.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject population, comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved. According to some embodiments, the geometric meanneutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and thegeometric mean neutralizing antibody titer for dengue serotype 4 (GMTDENV-4) when tested in at least 40, or at least 50, or at least 60subjects at day 180 or day 365 after at least a first administration ofsaid reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of GMT DENV-2:GMT DENV-4 of not more than 50, or notmore than 40, or not more than 30, or not more than 20. In some of theseembodiments, the ratio of GMT DENV-2:GMT DENV-1 is not more than 20, ornot more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof GMT DENV-2:GMT DENV-3 is not more than 20, or not more than 18, ornot more than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, comprising administering tothe subject at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of neutralizing antibody titersat day 180 or 365 after administration of said first unit dose to thesubject are achieved. According to some embodiments, the neutralizingantibody titer for dengue serotype 2 and the neutralizing antibody titerfor dengue serotype 4 at day 180 or day 365 after at least a firstadministration of the reconstituted unit dose of the invention asdescribed herein, and optionally a second administration of areconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for GMT DENV-4 ofnot more than 50, or not more than 40, or not more than 30, or not morethan 20. In some of these embodiments, the ratio of the neutralizingantibody titers of DENV-2:DENV-1 is not more than 20, or not more than18, or not more than 15 at day 180 or 365 after administration of saidfirst reconstituted unit dose, and/or the ratio of the neutralizingantibody titers of DENV-2:DENV-3 is not more than 20, or not more than18, or not more than 15 at day 180 or 365 after administration of saidfirst reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with the microneutralization testdisclosed herein, for example according to the method described inExample 2.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered by subcutaneous injection. According to some of theseembodiments the subcutaneous injection is administered to the arm,preferably to the deltoid region of the arm.

In certain embodiments the invention is directed to said uses, whereinone reconstituted unit dose of the invention as described isadministered by subcutaneous injection. According to some of theseembodiments, the subcutaneous injection is administered to the arm,preferably to the deltoid region of the arm.

In certain embodiments the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In one embodiment, the two unit doses are administeredwithin 12 months or more, or within six months, or within three months,such as at day 0 and day 90 or at day 1/0 and day 90. According to someof these embodiments a third unit dose of the invention as describedherein may be administered after the second administration. Such a thirdadministration may act as a booster and may be administered between 6 to12 months after the first administration, such as 12 months after thefirst administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments of the invention the subject is seronegative withrespect to all dengue serotypes.

In certain other embodiments of the invention the subject isseropositive with respect to at least one dengue serotype.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose is administered to the seronegative subjectby subcutaneous injection.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose is administered to a subject of unknownserostatus and/or wherein no test has been carried out to determinewhether the subject is seropositive or seronegative before the unit doseis administered.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered to a subject or subject population from a dengue endemicregion. In some of these embodiments, the subject or subject populationis from Singapore, Dominican Republic, Panama, Philippines, Colombia,Puerto Rico or Thailand, in particular from Singapore, DominicanRepublic, Panama, or Philippines. In other embodiments, the subject orsubject population is from a dengue non-endemic region. Such a subjectpopulation or subject may be vaccinated according to the invention inthe context of traveling to a dengue endemic region. In certainembodiments, the reconstituted unit dose of the invention as describedherein is administered subcutaneously to a subject or subject populationfrom a dengue endemic region or from a dengue non-endemic region.

In certain embodiments the invention is directed to said uses, whereinthe subject is of 2 to 60 years of age or more than 17 years, or morethan 18 years, or 18 to 60 years of age. In certain embodiments thesubject is 1 to 17 years of age, or less than 9 years of age, or lessthan 4 years of age or less than 2 years of age. In further embodiments,the subjects or subject population are adults of more than 21 years, or21 to 60 years, or 21 to 45 years of age. According to some of theseembodiments the subject is seronegative and from a dengue-endemicregion.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein is administered to apediatric subject or pediatric subject population of less than 2 yearsof age, preferably of 2 months to 2 years of age or 2 months to 1.5years of age or 2 months to 1 year of age. According to some of theseembodiments, the pediatric subject or pediatric subject population isseronegative and from a dengue endemic region.

In certain embodiments, the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously to a pediatric subject or pediatric subjectpopulation of less than 2 years of age, preferably of 2 months to 2years of age or 2 months to 1.5 years of age or 2 months to 1 year ofage. According to some of these embodiments, the pediatric subject orpediatric subject population is seronegative and from a dengue endemicregion.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in an elderly subject.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome(DSS) in an elderly subject.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered by subcutaneous injection. According to some of theseembodiments the subcutaneous injection is administered to the arm,preferably to the deltoid region of the arm.

In certain embodiments the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In one embodiment, the two unit doses are administeredwithin 12 months or more, or within six months, or within three months,such as at day 0/1 and day 90. According to some of these embodiments athird unit dose of the invention as described herein may be administeredafter the second administration. Such a third administration may act asa booster and may be administered between 6 to 12 months after the firstadministration, such as 12 months after the first administration, orlater than 12 month after the first administration, such as 12 months (1year) after the second administration or even 5 years or longer afterthe first or second administration.

In certain embodiments of the invention the elderly subject isseronegative with respect to all dengue serotypes.

In certain other embodiments of the invention the elderly subject isseropositive with respect to at least one dengue serotype.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose is administered to the seronegative elderlysubject by subcutaneous injection.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered to an elderly subject from a dengue endemic region. In someof these embodiments, the elderly subject is from Singapore, DominicanRepublic, Panama, Philippines, Colombia, Puerto Rico or Thailand, inparticular from Singapore, Dominican Republic, Panama, or Philippines.In other embodiments, the elderly subject is from a dengue non-endemicregion. Such an elderly subject may be vaccinated according to theinvention in the context of traveling to a dengue endemic region. Incertain embodiments, the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to an elderly subjectfrom a dengue endemic region or from a dengue non-endemic region.

In certain embodiments, the invention is directed to said uses, whereinthe elderly subject has at least one chronic condition or disease. Theat least one chronic condition or disease may be selected from diabetes,hypertension, allergies, previous strokes, ischemic heart disease,chronic renal impairment and chronic obstructive pulmonary disease.

In certain embodiments, the invention is directed to said uses, whereinthe elderly subject has an impaired immune system.

Method of Preventing Dengue Disease and Yellow Fever and Uses Method ofPrevention

The present invention is directed in part to a method of preventingdengue disease as well as yellow fever in a subject. Thus, in certainembodiments the invention is directed to a method of preventing denguedisease in a subject, comprising administering to the subject areconstituted unit dose of the invention as described herein, whereinthe method further comprises preventing yellow fever in the subject byconcomitant administration of a yellow fever vaccine, in particularYF-17D, to the subject.

The present invention is directed in part to a method of preventingdengue disease as well as yellow fever in a subject population. Thus, incertain embodiments the invention is directed to a method of preventingdengue disease in a subject population, comprising administering to thesubject population a reconstituted unit dose of the invention asdescribed herein, wherein the method further comprises preventing yellowfever in the subject population by concomitant administration of ayellow fever vaccine, in particular YF-17D, to the subject population.

The present invention is in part directed to said method for preventingdengue disease and yellow fever in a subject population comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved, and concomitantly administering a yellow fever vaccine, inparticular YF-17D, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease and yellow fever in a subject comprising administering tothe subject at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of neutralizing antibody titersat day 180 or 365 after administration of said first unit dose to thesubject are achieved, and concomitantly administering a yellow fevervaccine, in particular YF-17D, to the subject. According to someembodiments, the neutralizing antibody titer for dengue serotype 2 andthe neutralizing antibody titer for dengue serotype 4 at day 180 or day365 after at least a first administration of the reconstituted unit doseof the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said methods,wherein the unit dose of the invention as described herein and theyellow fever vaccine, in particular YF-17D, are administeredsimultaneously. In some of these embodiments the simultaneousadministration is on day 0 or day 90, preferably on day 0. In otherembodiments the administration of the unit dose of the invention asdescribed herein and the yellow fever vaccine, in particular YF-17D, aredone sequentially.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered and the yellow fever vaccine, in particular YF-17D, areadministered by subcutaneous injection. According to some embodiments,the subcutaneous injections are administered to the arm, preferably tothe deltoid region of the arm. According to some of these embodimentsthe subcutaneous injections of the unit dose of the invention asdescribed herein and yellow fever vaccine, in particular YF-17D, areadministered to different anatomical sites, such as to opposite arms, inparticular when the vaccines are administered simultaneously.

In certain embodiments the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 month or more, or within 6month, or within three months, such as at day 0/1 and day 90. Accordingto some of these embodiments a further third unit dose of the inventionas described herein is administered after the second. Such a thirdadministration may act as a booster and may be administered between 6 to12 months after the first administration, such as 12 months after thefirst administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and one dose of a yellow fever vaccine, in particular YF-17D, areadministered, in particular according to the following schedule

-   -   an administration of said yellow fever vaccine on day 0,    -   a first administration of the first reconstituted unit dose        after said yellow fever vaccine administration, such as 3 months        later and preferably on day 90, and    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 180.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and one dose of a yellow fever vaccine, in particular YF-17D, areadministered, in particular according to the following schedule

-   -   a first administration of the first reconstituted unit dose on        day 0,    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 90, and    -   an administration of said yellow fever vaccine after said second        administration of the reconstituted unit dose, such as 3 months        later and preferably on day 180.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and one dose of a yellow fever vaccine, in particular YF-17D, areadministered, in particular according to the following schedule

-   -   a simultaneous administration of the first reconstituted unit        dose and said yellow fever vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said simultaneous administration, such as 3 months later        and preferably on day 90.

In a preferred embodiment, the yellow fever vaccine and unit dose of theinvention as described herein are administered simultaneously on day 0or simultaneously on day 90.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saidmethods, wherein the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject or subjectpopulation and the yellow fever vaccine, in particular YF-17D vaccine,is administered subcutaneously to a subject or subject population, andwherein the subject or the subject population is seronegative withrespect to all dengue serotypes. In other embodiments, the subject orsubject population is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein and theyellow fever vaccine, in particular YF-17D, are administered to asubject or subject population from a dengue endemic region. In certainembodiments, the reconstituted unit dose of the invention as describedherein and the yellow fever vaccine, in particular YF-17D, areadministered subcutaneously to a subject or subject population from adengue endemic region. In other embodiments, the subject or subjectpopulation is from a dengue non-endemic region. Such a subjectpopulation or such a subject may be vaccinated according to the presentinvention in the context of traveling to a dengue endemic region andyellow fever endemic region.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinand of the yellow fever vaccine, in particular YF-17D, are administeredsubcutaneously to a subject or subject population of more than 17 years,or more than 18 years, or 18 to 60 years of age. In further embodiments,the subjects or subject population are adults of more than 21 years, or21 to 60 years, or 21 to 45 years of age. In some embodiments, thesubject or subject population is from a dengue endemic region. Inanother embodiment, the subject or subject population is from a denguenon-endemic region, preferably from a dengue non-endemic and yellowfever non-endemic region. According to some of these embodiments, thesubject or subject population are seronegative for all four dengueserotypes.

Unit Dose for Use in a Method of Prevention

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject, wherein the method also comprises the preventionof yellow fever in the subject with a yellow fever vaccine, inparticular YF-17D. In particular, the present invention is directed inpart to a unit dose of a dengue vaccine composition as described hereinand a yellow fever vaccine, in particular YF-17D, for use in a method ofpreventing dengue disease and yellow fever in a subject, respectively.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject population, wherein the method also comprises theprevention of yellow fever in the subject population with a yellow fevervaccine, in particular YF-17D. In particular, the present invention isdirected in part to a unit dose of a dengue vaccine composition asdescribed herein and a yellow fever vaccine, in particular YF-17D, foruse in a method of preventing dengue disease and yellow fever in asubject population, respectively.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the yellow fever vaccine, inparticular YF-17D, for use in a method of preventing dengue disease andyellow fever in a subject population, comprising administering to thesubject population at least a first reconstituted unit dose of theinvention as described herein, wherein certain ratios of geometric meanneutralizing antibody titers (GMTs) at day 180 or 365 afteradministration of said first unit dose to the subject population areachieved, and concomitantly administering a yellow fever vaccine, inparticular YF-17D, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the yellow fever vaccine, inparticular YF-17D, for use in a method of preventing dengue disease andyellow fever in a subject comprising administering to the subject atleast a first reconstituted unit dose of the invention as describedherein, wherein certain ratios of neutralizing antibody titers at day180 or 365 after administration of said first unit dose to the subjectare achieved, and concomitantly administering a yellow fever vaccine, inparticular YF-17D, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for DENV-4 of notmore than 50, or not more than 40, or not more than 30, or not more than20. In some of these embodiments, the ratio of the neutralizing antibodytiters of DENV-2:DENV-1 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of the neutralizing antibodytiters of DENV-2:DENV-3 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein said unit dose and said yellowfever vaccine are administered simultaneously. In some of theseembodiments, the simultaneous administration is on day 0 or day 90,preferably on day 0. In other embodiments, the administration of saidunit dose and said yellow fever vaccine, in particular YF-17D, are donesequentially.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein said unit dose is administeredby subcutaneous injection and wherein said yellow fever vaccine isadministered by subcutaneous injection. According to some embodiments,the subcutaneous injections are administered to the arm, preferably tothe deltoid region of the arm. According to some of these embodimentsthe subcutaneous injection of said unit dose and the subcutaneousinjection of said yellow fever vaccine are administered to differentanatomical sites, such as to opposite arms, in particular when thevaccines are administered simultaneously.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein two reconstituted unit dosesof the invention as described herein are administered. In someembodiments the two unit doses of the invention as described herein areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0/1 and day 90. According to some of theseembodiments a further third reconstituted unit dose of the invention asdescribed herein is administered after the second administration. Such athird administration may act as a booster and may be administeredbetween 6 to 12 months after the first administration, such as 12 monthsafter the first administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein two reconstituted unit dosesof the invention as described herein and one dose of the yellow fevervaccine are administered, in particular according to the followingschedule

-   -   an administration of said yellow fever vaccine on day 0,    -   a first administration of the first reconstituted unit dose        after said yellow fever vaccine administration, such as 3 months        later and preferably on day 90, and    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 180.

In another embodiment the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein two unit doses of theinvention as described herein and one dose of the yellow fever vaccineare administered, in particular according to the following schedule

-   -   a first administration of the first reconstituted unit on day 0,    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 90, and    -   an administration of said yellow fever vaccine after said second        administration of the reconstituted unit dose, such as 3 months        later and preferably on day 180.

In another embodiment the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein two unit doses of theinvention as described herein and one dose of the yellow fever vaccineare administered, in particular according to the following schedule

-   -   a simultaneous administration of the first reconstituted unit        dose and said yellow fever vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said simultaneous administration, such as 3 months later        and preferably on day 90.

In a preferred embodiment, the yellow fever vaccine and unit dose of theinvention as described herein are administered simultaneously on day 0or simultaneously on day 90.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein the subject or subjectpopulation is seronegative to all dengue serotypes. In certainembodiments, the invention is directed to the unit dose of the inventionas described herein and the yellow fever vaccine, in particular YF-17D,for said uses, wherein said unit dose is reconstituted and administeredsubcutaneously to a subject or subject population and said yellow fevervaccine is administered subcutaneously to a subject or subjectpopulation, and wherein the subject or the subject population isseronegative with respect to all dengue serotypes. In other embodiments,the subject or subject population is seropositive with respect to atleast one dengue serotype.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein said unit dose and said yellowfever vaccine are administered to a subject or subject population from adengue endemic region. In certain embodiments, the reconstituted unitdose of the invention as described herein and the yellow fever vaccine,in particular YF-17D, are administered subcutaneously to a subject orsubject population from a dengue endemic region. In other embodiments,the subject or subject population is from a dengue non-endemic region.Such a subject population or such a subject may be vaccinated accordingto the present invention in the context of traveling to a dengue endemicregion and yellow fever endemic region.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the yellow fever vaccine, inparticular YF-17D, for said uses, wherein the reconstituted unit dose ofthe invention as described herein and the yellow fever vaccine, inparticular YF-17D, are administered subcutaneously to a subject or asubject population of more than 17 years, or more than 18 years, or 18to 60 years of age. In some embodiments the subject or subjectpopulation is from a dengue endemic region. In another embodiment thesubject or subject population is from a dengue non-endemic region,preferably from a dengue non-endemic and yellow fever non-endemicregion. According to some of these embodiments, the subject or subjectpopulation is seronegative for all four dengue serotypes.

Use for the Manufacture of a Medicament in a Method of Prevention

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, further comprising the useof a yellow fever vaccine, in particular YF-17D, for the manufacture ofa medicament for preventing yellow fever in the subject. In particular,the present invention is directed in part to a use of a unit dose of adengue vaccine composition as described herein and a yellow fevervaccine, in particular YF-17D, for the manufacture of a medicament forpreventing dengue disease and yellow fever in a subject, respectively.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject population, furthercomprising the use of a yellow fever vaccine, in particular YF-17D, forthe manufacture of a medicament for preventing yellow fever in thesubject population. In particular, the present invention is directed inpart to a use of a unit dose of a dengue vaccine composition asdescribed herein and a yellow fever vaccine, in particular YF-17D, forthe manufacture of a medicament for preventing dengue disease and yellowfever in a subject population, respectively.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a yellow fever vaccine, inparticular YF-17D, for the manufacture of a medicament for preventingdengue disease and yellow fever in a subject population, comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved, and concomitantly administering a yellow fever vaccine, inparticular YF-17D, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a yellow fever vaccine, inparticular YF-17D, for the manufacture of a medicament for preventingdengue disease and yellow fever in a subject, comprising administeringto the subject at least a first reconstituted unit dose of the inventionas described herein, wherein certain ratios of neutralizing antibodytiters at day 180 or 365 after administration of said first unit dose tothe subject are achieved, and concomitantly administering a yellow fevervaccine, in particular YF-17D, to the subject. According to someembodiments, the neutralizing antibody titer for dengue serotype 2 andthe neutralizing antibody titer for dengue serotype 4 at day 180 or day365 after at least a first administration of the reconstituted unit doseof the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the yellow fevervaccine, in particular YF-17D, are administered simultaneously. In someof these embodiments the simultaneous administration is on day 0 or day90, preferably on day 0. In other embodiments the administration of theunit dose of the invention as described herein and the yellow fevervaccine, in particular YF-17D, are done sequentially.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein is reconstituted andadministered by subcutaneous injection and wherein the yellow fevervaccine, in particular YF-17D, is administered by subcutaneousinjection. According to some embodiments the injections are administeredto the arm, preferably to the deltoid region of the arm. According tosome of these embodiments the subcutaneous injection of the unit dose ofthe invention as described herein and of the yellow fever vaccine, inparticular YF-17D, are administered to different anatomical sites suchas to opposite arms, in particular when the vaccines are administeredsimultaneously.

In certain embodiments the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 months or more, or withinsix months, or within three months, such as at day 0/1 and day 90.According to some of these embodiments a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and one dose of ayellow fever vaccine, in particular YF-17D, are administered, inparticular according to the following schedule

-   -   an administration of said yellow fever vaccine on day 0,    -   a first administration of the first reconstituted unit dose        after said yellow fever vaccine administration, such as 3 months        later and preferably on day 90, and    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 180.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and one dose of ayellow fever vaccine, in particular YF-17D, are administered, inparticular according to the following schedule

-   -   a first administration of the first reconstituted unit dose on        day 0,    -   a second administration of the second reconstituted unit dose        said first administration of the reconstituted unit dose, such        as 3 months later and preferably on day 90, and    -   an administration of said yellow fever vaccine after said second        administration of the reconstituted unit dose, such as 3 months        later and preferably on day 180.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and one dose of ayellow fever vaccine, in particular YF-17D, are administered, inparticular according to the following schedule

-   -   a simultaneous administration of the first reconstituted unit        dose and said yellow fever vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said simultaneous administration, such as 3 months later        and preferably on day 90.

In certain embodiments, the invention is directed to said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saiduses, wherein the unit dose of the invention as described herein isreconstituted and administered subcutaneously to a subject or subjectpopulation and the yellow fever vaccine, in particular YF-17D, isadministered subcutaneously to a subject or subject population, andwherein the subject or the subject population is seronegative withrespect to all dengue serotypes. In other embodiments, the subject orsubject population is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the yellow fevervaccine, in particular YF-17D, are administered to a subject or subjectpopulation from a dengue endemic region. In certain embodiments, thereconstituted unit dose of the invention as described herein and theyellow fever vaccine, in particular YF-17D, are administeredsubcutaneously to a subject or subject population from a dengue endemicregion. In other embodiments, the subject or subject population is froma dengue non-endemic region. Such a subject population or such a subjectmay be vaccinated according to the present invention in the context oftraveling to a dengue endemic region and yellow fever endemic region.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein and theyellow fever vaccine, in particular YF-17D, are administeredsubcutaneously to a subject or subject population of more than 17 years,or more than 18 years, or 18 to 60 years of age. In further embodiments,the subjects or subject population are adults of more than 21 years, or21 to 60 years, or 21 to 45 years of age. In some embodiments, thesubject or subject population is from a dengue endemic region. Inanother embodiment, the subject or subject population is from a denguenon-endemic region, preferably from a dengue non-endemic and yellowfever non-endemic region. According to some of these embodiments, thesubject or subject population is seronegative for all four dengueserotypes.

Method of Preventing Dengue Disease and Hepatitis A and Uses Method ofPrevention

The present invention is directed in part to a method of preventingdengue disease as well as hepatitis A in a subject. Thus, in certainembodiments the invention is directed to a method of preventing denguedisease in a subject, comprising administering to the subject areconstituted unit dose of the invention as described herein, whereinthe method further comprises preventing hepatitis A in the subject byconcomitant administration of a hepatitis A vaccine, such as HAVRIX® orVAQTA®, to the subject.

The present invention is directed in part to a method of preventingdengue disease as well as hepatitis A in a subject population. Thus, incertain embodiments the invention is directed to a method of preventingdengue disease in a subject population, comprising administering to thesubject population a reconstituted unit dose of the invention asdescribed herein, wherein the method further comprises preventinghepatitis A in the subject population by concomitant administration of ahepatitis A vaccine, such as HAVRIX® or VAQTA®, to the subjectpopulation.

The present invention is in part directed to said method for preventingdengue disease and hepatitis A in a subject population comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved, and concomitantly administering a hepatitis A vaccine,such as HAVRIX® or VAQTA®, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or nor more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease and hepatitis A in a subject comprising administering tothe subject at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of neutralizing antibody titersat day 180 or 365 after administration of said first unit dose to thesubject are achieved, and concomitantly administering a hepatitis Avaccine, such as HAVRIX® or VAQTA®, to the subject. According to someembodiments, the neutralizing antibody titer for dengue serotype 2 andthe neutralizing antibody titer for dengue serotype 4 at day 180 or day365 after at least a first administration of the reconstituted unit doseof the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said methods,wherein the unit dose of the invention as described herein and thehepatitis A vaccine, such as HAVRIX® or VAQTA®, are administeredsimultaneously. In some of these embodiments, the simultaneousadministration is on day 0 or day 90, preferably on day 0. In otherembodiments, the administration of the unit dose of the invention asdescribed herein and the hepatitis A vaccine, such as HAVRIX® or VAQTA®,are done sequentially.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered by subcutaneous injection and wherein the hepatitis Avaccine, such as HAVRIX® or VAQTA®, is administered by intramuscularinjection. According to some embodiments, the injections areadministered to the arm, preferably to the deltoid region of the arm.According to some of these embodiments, the subcutaneous injection ofthe reconstituted unit dose of the invention as described herein and theintramuscular injection of the hepatitis A vaccine, such as HAVRIX® orVAQTA®, are administered to different anatomical sites, such as toopposite arms, in particular when the vaccines are administeredsimultaneously.

In certain embodiments, the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments, the two unit doses of the inventionas described herein are administered within 12 month or more, or within6 month, or within three months, such as at day 0/1 and day 90.According to some of these embodiments, a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments, the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and one dose of a hepatitis A vaccine, such as HAVRIX® or VAQTA®,are administered, in particular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and said hepatitis A vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saidmethods, wherein the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject or subjectpopulation and the hepatitis A vaccine, such as HAVRIX® or VAQTA®, isadministered intramuscularly to a subject or subject population, andwherein the subject or the subject population is seronegative withrespect to all dengue serotypes. In other embodiments, the subject orsubject population is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein and thehepatitis A vaccine, such as HAVRIX® or VAQTA®, are administered to asubject or subject population from a dengue endemic region. In certainembodiments, the reconstituted unit dose of the invention as describedherein is administered subcutaneously and the hepatitis A vaccine, suchas HAVRIX® or VAQTA®, is administered intramuscularly to a subject orsubject population from a dengue endemic region.

According to some embodiments, a second dose of a hepatitis A vaccine,such as HAVRIX® or VAQTA®, is administered. The second dose of thehepatitis A vaccine may be administered after the first administrationof the hepatitis A vaccine. Such a second administration may act as abooster and may be administered 9 months after the first administrationof the hepatitis A vaccine, such as on day 270.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously and wherein the hepatitis A vaccine, suchas HAVRIX® or VAQTA®, is administered intramuscularly to a subject orsubject population of more than 17 years, or more than 18 years, or 18to 60 years of age. In further embodiments, the subjects or subjectpopulation are adults of more than 21 years, or 21 to 60 years, or 21 to45 years of age. In some embodiments, the subject or subject populationis from a dengue endemic region. In another embodiment, the subject orsubject population is from a dengue non-endemic region, preferably froma dengue non-endemic and a hepatitis A non-endemic region. According tocertain embodiments, the subject or subject population is seronegativefor all four dengue serotypes.

Unit Dose for Use in a Method of Prevention

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject, wherein the method also comprises the preventionof hepatitis A in the subject with a hepatitis A vaccine, such asHAVRIX® or VAQTA®. In particular, the present invention is directed inpart to a unit dose of a dengue vaccine composition as described hereinand a hepatitis A vaccine, such as HAVRIX® or VAQTA®, for use in amethod of preventing dengue disease and hepatitis A in a subject,respectively.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject population, wherein the method also comprises theprevention of hepatitis A in the subject population with a hepatitis Avaccine, such as HAVRIX® or VAQTA®. In particular, the present inventionis directed in part to a unit dose of a dengue vaccine composition asdescribed herein and a hepatitis A vaccine, such as HAVRIX® or VAQTA®,for use in a method of preventing dengue disease and hepatitis A in asubject population, respectively.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the hepatitis A vaccine,such as HAVRIX® or VAQTA®, for use in a method of preventing denguedisease and hepatitis A in a subject population, comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved, and concomitantly administering a hepatitis A vaccine,such as HAVRIX® or VAQTA®, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the hepatitis A vaccine,such as HAVRIX® or VAQTA®, for use in a method of preventing denguedisease and hepatitis A in a subject comprising administering to thesubject a hepatitis A vaccine, such as HAVRIX® or VAQTA®, and at least afirst reconstituted unit dose of the invention as described herein,wherein certain ratios of neutralizing antibody titers at day 180 or 365after administration of said first unit dose to the subject areachieved, and concomitantly administering a hepatitis A vaccine, such asHAVRIX® or VAQTA®, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for DENV-4 of notmore than 50, or not more than 40, or not more than 30, or not more than20, wherein the method further comprises preventing hepatitis A in thesubject by concomitant administration of a hepatitis A vaccine, such asHAVRIX® or VAQTA®, to the subject. In some of these embodiments, theratio of the neutralizing antibody titers of DENV-2:DENV-1 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein said unit dose and saidhepatitis A vaccine are administered simultaneously. In some of theseembodiments, the simultaneous administration is on day 0 or day 90,preferably on day 0. In other embodiments, the administration of saidunit dose and said hepatitis A vaccine are done sequentially.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein said unit dose is administeredby subcutaneous injection and wherein said hepatitis A vaccine isadministered by intramuscular injection. According to some embodiments,the injections are administered to the arm, preferably to the deltoidregion of the arm. According to some of these embodiments, thesubcutaneous injection of said unit dose and the intramuscular injectionof said hepatitis A vaccine are administered to different anatomicalsites, such as to opposite arms, in particular when the vaccines areadministered simultaneously.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein two reconstituted unit dosesof the invention as described herein are administered. In someembodiments, the two unit doses of the invention as described herein areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0/1 and day 90. According to some of theseembodiments a further third reconstituted unit dose of the invention asdescribed herein is administered after the second administration. Such athird administration may act as a booster and may be administeredbetween 6 to 12 months after the first administration, such as 12 monthsafter the first administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein two reconstituted unit dosesof the invention as described herein and one dose of the hepatitis Avaccine are administered, in particular according to the followingschedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and a dose of said hepatitis A vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein the subject or subjectpopulation is seronegative to all dengue serotypes. In certainembodiments, the invention is directed to the unit dose of the inventionas described herein and the hepatitis A vaccine, such as HAVRIX® orVAQTA®, for said uses, wherein said unit dose is reconstituted andadministered subcutaneously to a subject or subject population and saidhepatitis A vaccine is administered intramuscularly to a subject orsubject population, and wherein the subject or the subject population isseronegative with respect to all dengue serotypes. In other embodiments,the subject or subject population is seropositive with respect to atleast one dengue serotype.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein said unit dose and saidhepatitis A vaccine are administered to a subject or subject populationfrom a dengue endemic region. In certain embodiments, the reconstitutedunit dose of the invention as described herein is administeredsubcutaneously and the hepatitis A vaccine, such as HAVRIX® or VAQTA®,is administered intramuscularly to a subject or subject population froma dengue endemic region.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein a further second dose of ahepatitis A vaccine, such as HAVRIX® or VAQTA®, is administered. Thesecond dose of the hepatitis A vaccine may be administered after thefirst administration of the hepatitis A vaccine. Such a secondadministration may act as a booster and may be administered 9 monthsafter the first administration of the hepatitis A vaccine, such as onday 270.

In some embodiments, the invention is directed to the unit dose of theinvention as described herein and the hepatitis A vaccine, such asHAVRIX® or VAQTA®, for said uses, wherein the reconstituted unit dose ofthe invention as described herein is administered subcutaneously and thehepatitis A vaccine, such as HAVRIX® or VAQTA®, is administeredintramuscularly to a subject or subject population of more than 17years, or more than 18 years, or 18 to 60 years of age. In furtherembodiments, the subjects or subject population are adults of more than21 years, or 21 to 60 years, or 21 to 45 years of age. In someembodiments, the subject or subject population is from a dengue endemicregion. In another embodiment, the subject or subject population is froma dengue non-endemic region, preferably from a dengue non-endemic and ahepatitis A non-endemic region. According to certain embodiments, thesubject or subject population is seronegative for all four dengueserotypes.

Use for the Manufacture of a Medicament in a Method of Prevention

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, further comprising the useof a hepatitis A vaccine, such as HAVRIX® or VAQTA®, for the manufactureof a medicament for preventing hepatitis A in the subject. Inparticular, the present invention is directed in part to a use of a unitdose of a dengue vaccine composition as described herein and a hepatitisA vaccine, such as HAVRIX® or VAQTA®, for the manufacture of amedicament for preventing dengue disease and hepatitis A in a subject,respectively.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject population, furthercomprising the use of a hepatitis A vaccine, such as HAVRIX® or VAQTA®,for the manufacture of a medicament for preventing hepatitis A in thesubject population. In particular, the present invention is directed inpart to a use of a unit dose of a dengue vaccine composition asdescribed herein and a hepatitis A vaccine, such as HAVRIX® or VAQTA®,for the manufacture of a medicament for preventing dengue disease andhepatitis A in a subject population, respectively.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a hepatitis A vaccine, such asHAVRIX® or VAQTA®, for the manufacture of a medicament for preventingdengue disease and hepatitis A in a subject population, comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved and concomitantly administering a hepatitis A vaccine, suchas HAVRIX® or VAQTA®, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a hepatitis A vaccine, such asHAVRIX® or VAQTA®, for the manufacture of a medicament for preventingdengue disease and hepatitis A in a subject, comprising administering tothe subject at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of neutralizing antibody titersat day 180 or 365 after administration of said first unit dose to thesubject are achieved and concomitantly administering a hepatitis Avaccine, such as HAVRIX® or VAQTA®, to the subject. According to someembodiments, the neutralizing antibody titer for dengue serotype 2 andthe neutralizing antibody titer for dengue serotype 4 at day 180 or day365 after at least a first administration of the reconstituted unit doseof the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the hepatitis Avaccine, such as HAVRIX® or VAQTA®, are administered simultaneously. Insome of these embodiments, the simultaneous administration is on day 0or day 90, preferably on day 0. In other embodiments, the administrationof the unit dose of the invention as described herein and the hepatitisA vaccine, such as HAVRIX® or VAQTA®, are done sequentially.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein is reconstituted andadministered by subcutaneous injection and wherein the hepatitis Avaccine, such as HAVRIX® or VAQTA®, is administered by intramuscularinjection. According to some embodiments, the injections areadministered to the arm, preferably to the deltoid region of the arm.According to some embodiments the subcutaneous injection of the unitdose of the invention as described herein and the intramuscularinjection of the hepatitis A vaccine, such as HAVRIX® or VAQTA®, areadministered to different anatomical sites, such as to opposite arms, inparticular when the vaccines are administered simultaneously.

In certain embodiments, the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments, the two unit doses of the inventionas described herein are administered within 12 months or more, or withinsix months, or within three months, such as at day 0/1 and day 90.According to some of these embodiments a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and one dose of ahepatitis A vaccine, such as HAVRIX® or VAQTA®, are administered, inparticular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and said hepatitis A vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.

In certain embodiments, the invention is directed to said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saiduses, wherein the unit dose of the invention as described herein isreconstituted and administered subcutaneously to a subject or subjectpopulation and the hepatitis A vaccine, such as HAVRIX® or VAQTA®, isadministered intramuscularly to a subject or subject population, andwherein the subject or the subject population is seronegative withrespect to all dengue serotypes. In other embodiments, the subject orsubject population is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the hepatitis Avaccine, such as HAVRIX® or VAQTA®, are administered to a subject orsubject population from a dengue endemic region. In certain embodiments,the reconstituted unit dose of the invention as described herein isadministered subcutaneously and the hepatitis A vaccine, such as HAVRIX®or VAQTA®, is administered intramuscularly to a subject or subjectpopulation from a dengue endemic region.

In certain embodiments, the invention is directed to said uses, whereina further second dose of a hepatitis A vaccine, such as HAVRIX® orVAQTA®, is administered. The second dose of the hepatitis A vaccine maybe administered after the first administration of the hepatitis Avaccine. Such a second administration may act as a booster and may beadministered 9 months after the first administration of the hepatitis Avaccine such as on day 270.

In certain embodiments, the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously and the hepatitis A vaccine, such as HAVRIX®or VAQTA®, is administered intramuscularly to a subject or subjectpopulation of more than 17 years, or more than 18 years, or 18 to 60years of age. In further embodiments, the subjects or subject populationare adults of more than 21 years, or 21 to 60 years, or 21 to 45 yearsof age. In some embodiments, the subject or subject population is from adengue endemic region. In another embodiment, the subject or subjectpopulation is from a dengue non-endemic region, preferably from a denguenon-endemic and hepatitis A non-endemic region. According to some ofthese embodiments, the subject or subject population is seronegative forall four dengue serotypes.

Method of Preventing Dengue Disease and HPV Associated Diseases and UsesMethod of Prevention

The present invention is directed in part to a method of preventingdengue disease as well as HPV-associated cancers or genital warts in asubject. Thus, in certain embodiments the invention is directed to amethod of preventing dengue disease in a subject, comprisingadministering to the subject a reconstituted unit dose of the inventionas described herein, wherein the method further comprises preventingHPV-associated cancers or genital warts in the subject by concomitantadministration of a HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9, to the subject.

The present invention is directed in part to a method of preventingdengue disease as well as HPV-associated cancers or genital warts in asubject population. Thus, in certain embodiments the invention isdirected to a method of preventing dengue disease in a subjectpopulation, comprising administering to the subject population areconstituted unit dose of the invention as described herein, whereinthe method further comprises preventing HPV-associated cancers orgenital warts in the subject population by concomitant administration ofa HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, tothe subject population.

The present invention is in part directed to said method for preventingdengue disease and HPV-associated cancers or genital warts in a subjectpopulation comprising administering to the subject population at least afirst reconstituted unit dose of the invention as described herein,wherein certain ratios of geometric mean neutralizing antibody titers(GMTs) at day 180 or 365 after administration of said first unit dose tothe subject population are achieved, and concomitantly administering aHPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, to thesubject population. According to some embodiments, the geometric meanneutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and thegeometric mean neutralizing antibody titer for dengue serotype 4 (GMTDENV-4) when tested in at least 40, or at least 50, or at least 60subjects at day 180 or day 365 after at least a first administration ofsaid reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of GMT DENV-2:GMT DENV-4 of not more than 50, or notmore than 40, or not more than 30, or not more than 20. In some of theseembodiments, the ratio of GMT DENV-2:GMT DENV-1 is not more than 20, ornot more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof GMT DENV-2:GMT DENV-3 is not more than 20, or not more than 18, ornot more than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease and HPV-associated cancers or genital warts in a subjectcomprising administering to the subject at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof neutralizing antibody titers at day 180 or 365 after administrationof said first unit dose to the subject are achieved, and concomitantlyadministering a HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for DENV-4 of notmore than 50, or not more than 40, or not more than 30, or not more than20. In some of these embodiments, the ratio of the neutralizing antibodytiters of DENV-2:DENV-1 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of the neutralizing antibodytiters of DENV-2:DENV-3 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said methods,wherein the unit dose of the invention as described herein and the HPVvaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, areadministered simultaneously. In some of these embodiments thesimultaneous administration is on day 0 or day 90, preferably on day 0.In other embodiments the administration of the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, are done sequentially.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered by subcutaneous injection and wherein the HPV vaccine,in particular a 9vHPV vaccine, such as GARDASIL® 9, is administered byintramuscular injection. According to some embodiments the injectionsare administered to the arm, preferably to the deltoid region of thearm. According to some of these embodiments the subcutaneous injectionof the unit dose of the invention as described herein and theintramuscular injection of the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, are administered to different anatomicalsites, such as to opposite arms, in particular when the vaccines areadministered simultaneously.

In certain embodiments the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 month or more, or within 6month, or within three months, such as at day 0/1 and day 90. Accordingto some of these embodiments a further third unit dose of the inventionas described herein is administered after the second. Such a thirdadministration may act as a booster and may be administered between 6 to12 months after the first administration, such as 12 months after thefirst administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and two doses of a HPV vaccine, in particular a 9vHPV vaccine,such as GARDASIL® 9, are administered, in particular according to thefollowing schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said HPV vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 month        later and preferably on day 90, and    -   a third administration of the second dose of said HPV vaccine        after said second administration of the reconstituted unit dose,        such as 3 month later and preferably on day 180.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saidmethods, wherein the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject or subjectpopulation and the HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9, is administered intramuscularly to a subject or subjectpopulation, and wherein the subject or the subject population isseronegative with respect to all dengue serotypes. In other embodiments,the subject or subject population is seropositive with respect to atleast one dengue serotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein and the HPVvaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, areadministered to a subject or subject population from a dengue endemicregion. In certain embodiments, the reconstituted unit dose of theinvention as described herein is administered subcutaneously and the HPVvaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, isadministered intramuscularly to a subject or subject population from adengue endemic region.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously and wherein the HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9, is administeredintramuscularly to a subject or subject population of 9 to 26 years ofage, preferably 9 to 15 years of age. In some embodiments the subject orsubject population is from a dengue endemic region. According to some ofthese embodiments, the subject or subject population is female.According to some of these embodiments, the subject or subjectpopulation is seronegative for all four dengue serotypes.

In a preferred embodiment the invention is directed to a method ofpreventing dengue disease and HPV-associated cancers or genital warts ina subject comprising administering to the subject a subcutaneousinjection of the reconstituted unit dose of the invention as describedherein and an intramuscular injection of the HPV vaccine, in particulara 9vHPV vaccine, such as GARDASIL® 9, according to the above describedadministration schedule, wherein the subject is a female subject of 9 to15 years of age from a dengue endemic region.

Unit Dose for Use in a Method of Prevention

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject, wherein the method also comprises the preventionof HPV-associated cancers or genital warts in the subject with a HPVvaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9. Inparticular, the present invention is directed in part to a unit dose ofa dengue vaccine composition as described herein and a HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9, for use in a method ofpreventing dengue disease and HPV-associated cancers or genital warts ina subject, respectively.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject population, wherein the method also comprises theprevention of HPV-associated cancers or genital warts in the subjectpopulation with a HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9. In particular, the present invention is directed in part toa unit dose of a dengue vaccine composition as described herein and aHPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, for usein a method of preventing dengue disease and HPV-associated cancers orgenital warts in a subject population, respectively.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9, for use in a method ofpreventing dengue disease and HPV-associated cancers or genital warts ina subject population, comprising administering to the subject populationat least a first reconstituted unit dose of the invention as describedherein, wherein certain ratios of geometric mean neutralizing antibodytiters (GMTs) at day 180 or 365 after administration of said first unitdose to the subject population are achieved, and concomitantlyadministering a HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9, to the subject population. According to some embodiments,the geometric mean neutralizing antibody titer for dengue serotype 2(GMT DENV-2) and the geometric mean neutralizing antibody titer fordengue serotype 4 (GMT DENV-4) when tested in at least 40, or at least50, or at least 60 subjects at day 180 or day 365 after at least a firstadministration of said reconstituted unit dose of the invention asdescribed herein, and optionally a second administration of areconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9, for use in a method ofpreventing dengue disease and HPV-associated cancers or genital warts ina subject comprising administering to the subject at least a firstreconstituted unit dose of the invention as described herein, whereincertain ratios of neutralizing antibody titers at day 180 or 365 afteradministration of said first unit dose to the subject are achieved, andconcomitantly administering a HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, to the subject. According to someembodiments, the neutralizing antibody titer for dengue serotype 2 andthe neutralizing antibody titer for dengue serotype 4 at day 180 or day365 after at least a first administration of the reconstituted unit doseof the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, for said uses, wherein said unit dose andsaid HPV vaccine are administered simultaneously. In some of theseembodiments the simultaneous administration is on day 0 or day 90,preferably on day 0. In other embodiments the administration of saidunit dose and said HPV vaccine are done sequentially.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, for said uses, wherein said unit dose isadministered by subcutaneous injection and wherein said HPV vaccine isadministered by intramuscular injection. According to some embodimentsthe injections are administered to the arm, preferably to the deltoidregion of the arm. According to some of these embodiments thesubcutaneous injection of said unit dose and the intramuscular injectionof said HPV vaccine are administered to different anatomical sites, suchas to opposite arms, in particular when the vaccines are administeredsimultaneously.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, for said uses, wherein two reconstitutedunit doses of the invention as described herein are administered. Insome embodiments the two unit doses of the invention as described hereinare administered within 12 months or more, or within six months, orwithin three months, such as at day 0/1 and day 90. According to some ofthese embodiments a further third reconstituted unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, for said uses, wherein two reconstitutedunit doses of the invention as described herein and two doses of the HPVvaccine are administered, in particular according to the followingschedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said HPV vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 month        later and preferably on day 90, and    -   a third administration of the second dose of said HPV vaccine        after said second administration of the reconstituted unit dose,        such as 3 month later and preferably on day 180.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the HPV vaccine, in particular a9vHPV vaccine, such as GARDASIL® 9, for said uses, wherein the subjector subject population is seronegative to all dengue serotypes. Incertain embodiments, the invention is directed to the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, for said uses, wherein said unit dose isreconstituted and administered subcutaneously to a subject or subjectpopulation and said HPV vaccine is administered intramuscularly to asubject or subject population, and wherein the subject or the subjectpopulation is seronegative with respect to all dengue serotypes. Inother embodiments, the subject or subject population is seropositivewith respect to at least one dengue serotype.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the HPV vaccine, in particular a9vHPV vaccine, such as GARDASIL® 9, for said uses, wherein said unitdose and said HPV vaccine are administered to a subject or subjectpopulation from a dengue endemic region. In certain embodiments, thereconstituted unit dose of the invention as described herein isadministered subcutaneously and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, is administered intramuscularly to asubject or subject population from a dengue endemic region.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, for said uses, wherein the reconstitutedunit dose of the invention as described herein is administeredsubcutaneously and wherein the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, is administered intramuscular to a subjector subject population of 9 to 25 years of age, preferably of 9 to 15years of age. In some embodiments the subject or subject population isfrom a dengue endemic region. According to some of these embodiments,the subject or subject population is female. According to some of theseembodiments, the subject or subject population is seronegative for allfour dengue serotypes.

In a preferred embodiment the invention is directed to the unit dose ofthe invention as described herein and the HPV vaccine, in particular a9vHPV vaccine, such as GARDASIL® 9, for use in a method of preventingdengue disease and HPV-associated cancers or genital warts in a subjectcomprising administering to the subject a subcutaneous injection of thereconstituted unit dose of the invention as described herein and anintramuscular injection of the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, according to the above describedadministration schedule, wherein the subject is a female subject of 9 to15 years of age from a dengue endemic region.

Use for the Manufacture of a Medicament in a Method of Prevention

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, further comprising the useof a HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9,for the manufacture of a medicament for preventing HPV-associatedcancers or genital warts in the subject. In particular, the presentinvention is directed in part to a use of a unit dose of a denguevaccine composition as described herein and a HPV vaccine, in particulara 9vHPV vaccine, such as GARDASIL® 9, for the manufacture of amedicament for preventing dengue disease and HPV-associated cancers orgenital warts in a subject, respectively.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject population, furthercomprising the use of a HPV vaccine, in particular a 9vHPV vaccine, suchas GARDASIL® 9, for the manufacture of a medicament for preventingHPV-associated cancers or genital warts in the subject population. Inparticular, the present invention is directed in part to a use of a unitdose of a dengue vaccine composition as described herein and a HPVvaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, for themanufacture of a medicament for preventing dengue disease andHPV-associated cancers or genital warts in a subject population,respectively.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a HPV vaccine, in particular a9vHPV vaccine, such as GARDASIL® 9, for the manufacture of a medicamentfor preventing dengue disease and HPV-associated cancers or genitalwarts in a subject population, comprising administering to the subjectpopulation at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of geometric mean neutralizingantibody titers (GMTs) at day 180 or 365 after administration of saidfirst unit dose to the subject population are achieved, andconcomitantly administering a HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, to the subject population. According tosome embodiments, the geometric mean neutralizing antibody titer fordengue serotype 2 (GMT DENV-2) and the geometric mean neutralizingantibody titer for dengue serotype 4 (GMT DENV-4) when tested in atleast 40, or at least 50, or at least 60 subjects at day 180 or day 365after at least a first administration of said reconstituted unit dose ofthe invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of GMT DENV-2:GMT DENV-4 of not more than 50, or not more than 40,or not more than 30, or not more than 20. In some of these embodiments,the ratio of GMT DENV-2:GMT DENV-1 is not more than 20, or not more than18, or not more than 15 at day 180 or 365 after administration of saidfirst reconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3is not more than 20, or not more than 18, or not more than 15 at day 180or 365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a HPV vaccine, in particular a9vHPV vaccine, such as GARDASIL® 9, for the manufacture of a medicamentfor preventing dengue disease and HPV-associated cancers or genitalwarts in a subject, comprising administering to the subject at least afirst reconstituted unit dose of the invention as described herein,wherein certain ratios of neutralizing antibody titers at day 180 or 365after administration of said first unit dose to the subject areachieved, and concomitantly administering a HPV vaccine, in particular a9vHPV vaccine, such as GARDASIL® 9, to the subject. According to someembodiments, the neutralizing antibody titer for dengue serotype 2 andthe neutralizing antibody titer for dengue serotype 4 at day 180 or day365 after at least a first administration of the reconstituted unit doseof the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the HPV vaccine,in particular a 9vHPV vaccine, such as GARDASIL® 9, are administeredsimultaneously. In some of these embodiments the simultaneousadministration is on day 0 or day 90, preferably on day 0. In otherembodiments the administration of the unit dose of the invention asdescribed herein and the HPV vaccine, in particular a 9vHPV vaccine,such as GARDASIL® 9, are done sequentially.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein is reconstituted andadministered by subcutaneous injection and wherein the HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9, is administered byintramuscular injection. According to some embodiments the injectionsare administered to the arm, preferably to the deltoid region of thearm. According to some of these embodiments the subcutaneous injectionof the unit dose of the invention as described herein and theintramuscular injection of the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, are administered to different anatomicalsites such as to opposite arms, in particular when the vaccines areadministered simultaneously.

In certain embodiments the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 months or more, or withinsix months, or within three months, such as at day 0/1 and day 90.According to some of these embodiments a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and two doses of aHPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, areadministered, in particular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said HPV vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said simultaneous administration, such as 3 month later        and preferably on day 90, and    -   a third administration of the second dose of said HPV vaccine        after said second administration of the reconstituted unit dose,        such as 3 month later and preferably on day 180.

In certain embodiments, the invention is directed to said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saiduses, wherein the unit dose of the invention as described herein isreconstituted and administered subcutaneously to a subject or subjectpopulation and the HPV vaccine, in particular a 9vHPV vaccine, such asGARDASIL® 9, is administered intramuscularly to a subject or subjectpopulation, and wherein the subject or the subject population isseronegative with respect to all dengue serotypes. In other embodiments,the subject or subject population is seropositive with respect to atleast one dengue serotype.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the HPV vaccine,in particular a 9vHPV vaccine, such as GARDASIL® 9, are administered toa subject or subject population from a dengue endemic region. In certainembodiments, the reconstituted unit dose of the invention as describedherein is administered subcutaneously and the HPV vaccine, in particulara 9vHPV vaccine, such as GARDASIL® 9, is administered intramuscularly toa subject or subject population from a dengue endemic region.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously and the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, is administered intramuscularly to asubject or subject population of 9 years to 25 years of age, preferablyof 9 to 15 years of age. In some embodiments the subject or subjectpopulation is from a dengue endemic region. According to some of theseembodiments, the subject or subject population is female. According tosome of these embodiments, the subject or subject population isseronegative for all four dengue serotypes.

In a preferred embodiment the invention is directed to the use of a unitdose of a dengue vaccine composition as described herein and a HPVvaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, for themanufacture of a medicament for preventing dengue disease andHPV-associated cancers or genital warts in a subject comprisingadministering to the subject a subcutaneous injection of thereconstituted unit dose of the invention as described herein and anintramuscular injection of the HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, according to the above describedadministration schedule, wherein the subject is a female subject of 9 to15 years of age from a dengue endemic region.

Method of Preventing Dengue Disease and Measles, Mumps and Rubella andUses Method of Prevention

The present invention is directed in part to a method of preventingdengue disease as well as measles, mumps and rubella in a subject. Thus,in certain embodiments the invention is directed to a method ofpreventing dengue disease in a subject, comprising administering to thesubject a reconstituted unit dose of the invention as described herein,wherein the method further comprises preventing measles, mumps andrubella in the subject by concomitant administration of a MMR vaccine,such as M-M-R® II, to the subject.

The present invention is directed in part to a method of preventingdengue disease as well as measles, mumps and rubella in a subjectpopulation. Thus, in certain embodiments the invention is directed to amethod of preventing dengue disease in a subject population, comprisingadministering to the subject population a reconstituted unit dose of theinvention as described herein, wherein the method further comprisespreventing measles, mumps and rubella in the subject population byconcomitant administration of a MMR vaccine, such as M-M-R® II, to thesubject population.

The present invention is in part directed to said method for preventingdengue disease and measles, mumps and rubella in a subject populationcomprising administering to the subject population at least a firstreconstituted unit dose of the invention as described herein, whereincertain ratios of geometric mean neutralizing antibody titers (GMTs) atday 180 or 365 after administration of said first unit dose to thesubject population are achieved, and concomitantly administering a MMRvaccine, such as M-M-R® II, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease and measles, mumps and rubella in a subject comprisingadministering to the subject at least a first reconstituted unit dose ofthe invention as described herein, wherein certain ratios ofneutralizing antibody titers at day 180 or 365 after administration ofsaid first unit dose to the subject are achieved, and concomitantlyadministering a MMR vaccine, such as M-M-R® II, to the subject.According to some embodiments, the neutralizing antibody titer fordengue serotype 2 and the neutralizing antibody titer for dengueserotype 4 at day 180 or day 365 after at least a first administrationof the reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of neutralizing antibody titer for DENV-2:neutralizingantibody titer for DENV-4 of not more than 50, or not more than 40, ornot more than 30, or not more than 20. In some of these embodiments, theratio of the neutralizing antibody titers of DENV-2:DENV-1 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said methods,wherein the unit dose of the invention as described herein and the MMRvaccine, such as M-M-R® II, are administered simultaneously. In some ofthese embodiments the simultaneous administration is on day 0 or day 90,preferably on day 0. In other embodiments the administration of the unitdose of the invention as described herein and the MMR vaccine, such asM-M-R® II, are done sequentially.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered by subcutaneous injection and wherein the MMR vaccine,such as M-M-R® II, is administered by subcutaneous injection. Accordingto some embodiments the injections are administered to the arm,preferably to the deltoid region of the arm. According to some of theseembodiments the subcutaneous injection of the unit dose of the inventionas described herein and the subcutaneous injection of the MMR vaccine,such as M-M-R® II, are administered to different anatomical sites, suchas to opposite arms, in particular when the vaccines are administeredsimultaneously.

In certain embodiments the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 month or more, or within 6month, or within three months, such as at day 0/1 and day 90. Accordingto some of these embodiments a further third unit dose of the inventionas described herein is administered after the second. Such a thirdadministration may act as a booster and may be administered between 6 to12 months after the first administration, such as 12 months after thefirst administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and two doses of a MMR vaccine, such as M-M-R® II, areadministered, in particular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said MMR vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90, and    -   a third administration of the second dose of said MMR vaccine        after said second administration of the second reconstituted        unit dose, such as 3 to 6 years after said first simultaneous        administration.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saidmethods, wherein the reconstituted unit dose of the invention asdescribed herein and the MMR vaccine, such as M-M-R® II, areadministered subcutaneously to a subject or subject population, andwherein the subject or the subject population is seronegative withrespect to all dengue serotypes. In other embodiments, the subject orsubject population is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein and the MMRvaccine, such as M-M-R® II, are administered to a subject or subjectpopulation from a dengue endemic region. In certain embodiments, thereconstituted unit dose of the invention as described herein and the MMRvaccine, such as M-M-R® II, are administered subcutaneously to a subjector subject population from a dengue endemic region.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinand wherein the MMR vaccine, such as M-M-R® II, are administeredsubcutaneously to a subject or subject population of 1 to 10 years ofage, preferably 2 months to 7 years of age. In some embodiments thesubject or subject population is from a dengue endemic region. Inanother embodiment, the subject or subject population is from a denguenon-endemic region. According to some of these embodiments, the subjector subject population is seronegative for all four dengue serotypes.

Unit Dose for Use in a Method of Prevention

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject, wherein the method also comprises the preventionof measles, mumps and rubella in the subject with a MMR vaccine, such asM-M-R® II. In particular, the present invention is directed in part to aunit dose of a dengue vaccine composition as described herein and a MMRvaccine, such as M-M-R® II, for use in a method of preventing denguedisease and measles, mumps and rubella in a subject, respectively.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject population, wherein the method also comprises theprevention of measles, mumps and rubella in the subject population witha MMR vaccine, such as M-M-R® II. In particular, the present inventionis directed in part to a unit dose of a dengue vaccine composition asdescribed herein and a MMR vaccine, such as M-M-R® II, for use in amethod of preventing dengue disease and measles, mumps and rubella in asubject population, respectively.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the MMR vaccine, such asM-M-R® II, for use in a method of preventing dengue disease and measles,mumps and rubella in a subject population, comprising administering tothe subject population at least a first reconstituted unit dose of theinvention as described herein, wherein certain ratios of geometric meanneutralizing antibody titers (GMTs) at day 180 or 365 afteradministration of said first unit dose to the subject population areachieved, and concomitantly administering a MMR vaccine, such as M-M-R®II, to the subject population. According to some embodiments, thegeometric mean neutralizing antibody titer for dengue serotype 2 (GMTDENV-2) and the geometric mean neutralizing antibody titer for dengueserotype 4 (GMT DENV-4) when tested in at least 40, or at least 50, orat least 60 subjects at day 180 or day 365 after at least a firstadministration of said reconstituted unit dose of the invention asdescribed herein, and optionally a second administration of areconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the MMR vaccine, such asM-M-R® II, for use in a method of preventing dengue disease and measles,mumps and rubella in a subject comprising administering to the subjectat least a first reconstituted unit dose of the invention as describedherein, wherein certain ratios of neutralizing antibody titers at day180 or 365 after administration of said first unit dose to the subjectare achieved, and concomitantly administering a MMR vaccine, such asM-M-R® II, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for DENV-4 of notmore than 50, or not more than 40, or not more than 30, or not more than20. In some of these embodiments, the ratio of the neutralizing antibodytiters of DENV-2:DENV-1 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of the neutralizing antibodytiters of DENV-2:DENV-3 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the MMR vaccine, such as M-M-R® II,for said uses, wherein said unit dose and said MMR vaccine areadministered simultaneously. In some of these embodiments thesimultaneous administration is on day 0 or day 90, preferably on day 0.In other embodiments the administration of said unit dose and said MMRvaccine are done sequentially.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the MMR vaccine, such as M-M-R® II,for said uses, wherein said unit dose is administered by subcutaneousinjection and wherein said MMR vaccine is administered by subcutaneousinjection. According to some embodiments the injections are administeredto the arm, preferably to the deltoid region of the arm. According tosome of these embodiments the subcutaneous injection of said unit doseand the subcutaneous injection of said MMR vaccine are administered todifferent anatomical sites, such as to opposite arms, in particular whenthe vaccines are administered simultaneously.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the MMR vaccine, such as M-M-R® II,for said uses, wherein two reconstituted unit doses of the invention asdescribed herein are administered. In some embodiments the two unitdoses of the invention as described herein are administered within 12months or more, or within six months, or within three months, such as atday 0/1 and day 90. According to some of these embodiments a furtherthird reconstituted unit dose of the invention as described herein isadministered after the second administration. Such a thirdadministration may act as a booster and may be administered between 6 to12 months after the first administration, such as 12 months after thefirst administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the MMR vaccine, such as M-M-R® II,for said uses, wherein two reconstituted unit doses of the invention asdescribed herein and two doses of the MMR vaccine are administered, inparticular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said MMR vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90, and    -   a third administration of the second dose of said MMR vaccine        after said second administration of the second reconstituted        unit dose, such as 3 to 6 years after said first simultaneous        administration.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the MMR vaccine, such as M-M-R®II, for said uses, wherein the subject or subject population isseronegative to all dengue serotypes. In certain embodiments, theinvention is directed to the unit dose of the invention as describedherein and the MMR vaccine, such as M-M-R® II, for said uses, whereinsaid unit dose is reconstituted and administered subcutaneously to asubject or subject population and said MMR vaccine is administeredsubcutaneously to a subject or subject population, and wherein thesubject or the subject population is seronegative with respect to alldengue serotypes. In other embodiments, the subject or subjectpopulation is seropositive with respect to at least one dengue serotype.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the MMR vaccine, such as M-M-R®II, for said uses, wherein said unit dose and said MMR vaccine areadministered to a subject or subject population from a dengue endemicregion. In certain embodiments, the reconstituted unit dose of theinvention as described herein and the MMR vaccine, such as M-M-R® II,are administered subcutaneously to a subject or subject population froma dengue endemic region.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the MMR vaccine, such as M-M-R® II,for said uses, wherein the reconstituted unit dose of the invention asdescribed herein and the MMR vaccine, such as M-M-R® II, areadministered subcutaneously to a subject or subject population of 1 to10 years of age, preferably of 2 months to 7 years of age. In someembodiments, the subject or subject population is from a dengue endemicregion. In another embodiment, the subject or subject population is froma dengue non-endemic region. According to some of these embodiments, thesubject or subject population is seronegative for all four dengueserotypes.

Use for the Manufacture of a Medicament in a Method of Prevention

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, further comprising the useof a MMR vaccine, such as M-M-R® II, for the manufacture of a medicamentfor preventing measles, mumps and rubella in the subject. In particular,the present invention is directed in part to a use of a unit dose of adengue vaccine composition as described herein and a MMR vaccine, suchas M-M-R® II, for the manufacture of a medicament for preventing denguedisease and measles, mumps and rubella in a subject, respectively.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject population, furthercomprising the use of a MMR vaccine, such as M-M-R® II, for themanufacture of a medicament for preventing measles, mumps and rubella inthe subject population. In particular, the present invention is directedin part to a use of a unit dose of a dengue vaccine composition asdescribed herein and a MMR vaccine, such as M-M-R® II, for themanufacture of a medicament for preventing dengue disease and measles,mumps and rubella in a subject population, respectively.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a MMR vaccine, such as M-M-R® II,for the manufacture of a medicament for preventing dengue disease andmeasles, mumps and rubella in a subject population, comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved, and concomitantly administering a MMR vaccine, such asM-M-R® II, to the subject population. According to some embodiments, thegeometric mean neutralizing antibody titer for dengue serotype 2 (GMTDENV-2) and the geometric mean neutralizing antibody titer for dengueserotype 4 (GMT DENV-4) when tested in at least 40, or at least 50, orat least 60 subjects at day 180 or day 365 after at least a firstadministration of said reconstituted unit dose of the invention asdescribed herein, and optionally a second administration of areconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a MMR vaccine, such as M-M-R® II,for the manufacture of a medicament for preventing dengue disease andmeasles, mumps and rubella in a subject, comprising administering to thesubject at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of neutralizing antibody titersat day 180 or 365 after administration of said first unit dose to thesubject are achieved, and concomitantly administering a MMR vaccine,such as M-M-R® II, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for DENV-4 of notmore than 50, or not more than 40, or not more than 30, or not more than20. In some of these embodiments, the ratio of the neutralizing antibodytiters of DENV-2:DENV-1 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of the neutralizing antibodytiters of DENV-2:DENV-3 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the MMR vaccine,such as M-M-R® II, are administered simultaneously. In some of theseembodiments the simultaneous administration is on day 0 or day 90,preferably on day 0. In other embodiments the administration of the unitdose of the invention as described herein and the MMR vaccine, such asM-M-R® II, are done sequentially.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein is reconstituted andadministered by subcutaneous injection and wherein the MMR vaccine, suchas M-M-R® II, is administered by subcutaneous injection. According tosome embodiments the injections are administered to the arm, preferablyto the deltoid region of the arm. According to some of these embodimentsthe subcutaneous injection of the unit dose of the invention asdescribed herein and the subcutaneous injection of the MMR vaccine, suchas M-M-R® II, are administered to different anatomical sites such as toopposite arms, in particular when the vaccines are administeredsimultaneously.

In certain embodiments the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 months or more, or withinsix months, or within three months, such as at day 0/1 and day 90.According to some of these embodiments a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and two doses of aMMR vaccine, such as M-M-R® II, are administered, in particularaccording to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said MMR vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90, and    -   a third administration of the second dose of said MMR vaccine        after said second administration of the second reconstituted        unit dose, such as 3 to 6 years after said first simultaneous        administration.

In certain embodiments, the invention is directed to said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saiduses, wherein the unit dose of the invention as described herein isreconstituted and administered subcutaneously to a subject or subjectpopulation and the MMR vaccine, such as M-M-R® II, is administeredsubcutaneously to a subject or subject population, and wherein thesubject or the subject population is seronegative with respect to alldengue serotypes. In other embodiments, the subject or subjectpopulation is seropositive with respect to at least one dengue serotype.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the MMR vaccine,such as M-M-R® II, are administered to a subject or subject populationfrom a dengue endemic region. In certain embodiments, the reconstitutedunit dose of the invention as described herein and the MMR vaccine, suchas M-M-R® II, are administered subcutaneously to a subject or subjectpopulation from a dengue endemic region.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein and theMMR vaccine, such as M-M-R® II, are administered subcutaneously to asubject or subject population of 1 to 10 years of age, preferably of 2months to 7 years of age. In some embodiments, the subject or subjectpopulation is from a dengue endemic region. In another embodiment, thesubject or subject population is from a dengue non-endemic region.According to some of these embodiments, the subject or subjectpopulation is seronegative for all four dengue serotypes.

Method of Preventing Dengue Disease and Tetanus, Diphtheria, andPertussis and Uses Method of Prevention

The present invention is directed in part to a method of preventingdengue disease, as well as tetanus, diphtheria, and pertussis in asubject. Thus, in certain embodiments the invention is directed to amethod of preventing dengue disease in a subject, comprisingadministering to the subject a reconstituted unit dose of the inventionas described herein, wherein the method further comprises preventingtetanus, diphtheria, and pertussis in the subject by concomitantadministration of a Tdap vaccine, in particular a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, to the subject.

The present invention is directed in part to a method of preventingdengue disease, as well as tetanus, diphtheria, and pertussis in asubject population. Thus, in certain embodiments the invention isdirected to a method of preventing dengue disease, in a subjectpopulation, comprising administering to the subject population areconstituted unit dose of the invention as described herein, whereinthe method further comprises preventing tetanus, diphtheria, andpertussis in the subject population by concomitant administration of aTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, to the subject population.

The present invention is in part directed to said method for preventingdengue disease and tetanus, diphtheria, and pertussis in a subjectpopulation comprising administering to the subject population at least afirst reconstituted unit dose of the invention as described herein,wherein certain ratios of geometric mean neutralizing antibody titers(GMTs) at day 180 or 365 after administration of said first unit dose tothe subject population are achieved, and concomitantly administering aTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, to the subject population. According to some embodiments, thegeometric mean neutralizing antibody titer for dengue serotype 2 (GMTDENV-2) and the geometric mean neutralizing antibody titer for dengueserotype 4 (GMT DENV-4) when tested in at least 40, or at least 50, orat least 60 subjects at day 180 or day 365 after at least a firstadministration of said reconstituted unit dose of the invention asdescribed herein, and optionally a second administration of areconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or nor more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease and tetanus, diphtheria, and pertussis in a subjectcomprising administering to the subject at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof neutralizing antibody titers at day 180 or 365 after administrationof said first unit dose to the subject are achieved, and concomitantlyadministering a Tdap vaccine, in particular a combined tetanus toxoid,reduced diphtheria toxoid and acellular pertussis (adsorbed) vaccine,such as BOOSTRIX®, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for GMT DENV-4 ofnot more than 50, or not more than 40, or not more than 30, or not morethan 20. In some of these embodiments, the ratio of the neutralizingantibody titers of DENV-2:DENV-1 is not more than 20, or not more than18, or not more than 15 at day 180 or 365 after administration of saidfirst reconstituted unit dose, and/or the ratio of the neutralizingantibody titers of DENV-2:DENV-3 is not more than 20, or not more than18, or not more than 15 at day 180 or 365 after administration of saidfirst reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said methods,wherein the unit dose of the invention as described herein and the Tdapvaccine, in particular a combined tetanus toxoid, reduced diphtheriatoxoid and acellular pertussis (adsorbed) vaccine, such as BOOSTRIX®,are administered simultaneously. In some of these embodiments, thesimultaneous administration is on day 0 or day 90, preferably on day 0.In other embodiments, the administration of the unit dose of theinvention as described herein and the Tdap vaccine, in particularcombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, are done sequentially.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered by subcutaneous injection and wherein the Tdap vaccine,in particular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, isadministered by intramuscular injection. According to some embodiments,the injections are administered to the arm, preferably to the deltoidregion of the arm. According to some of these embodiments, thesubcutaneous injection of the unit dose of the invention as describedherein and the intramuscular injection of the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, areadministered to different anatomical sites, such as to opposite arms, inparticular when the vaccines are administered simultaneously.

In certain embodiments, the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments, the two unit doses of the inventionas described herein are administered within 12 months or more, or within6 months, or within 3 months, such as at day 0/1 and day 90. Accordingto some of these embodiments a further third unit dose of the inventionas described herein is administered after the second administration.Such a third administration may act as a booster and may be administeredbetween 6 to 12 months after the first administration, such as 12 monthsafter the first administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments, the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and one dose of a Tdap vaccine, in particular a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, are administered, in particular according tothe following schedule:

-   -   a first simultaneous administration of the first reconstituted        unit dose and said Tdap vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saidmethods, wherein the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject or subjectpopulation and the Tdap vaccine, in particular a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, is administered intramuscularly to a subjector subject population, and wherein the subject or the subject populationis seronegative with respect to all dengue serotypes. In otherembodiments, the subject or subject population is seropositive withrespect to at least one dengue serotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein and the Tdapvaccine, in particular a combined tetanus toxoid, reduced diphtheriatoxoid and acellular pertussis (adsorbed) vaccine, such as BOOSTRIX®,are administered to a subject or subject population from a dengueendemic region. In certain embodiments, the reconstituted unit dose ofthe invention as described herein is administered subcutaneously and theTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, is administered intramuscularly to a subject or subjectpopulation from a dengue endemic region.

In certain embodiments, the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously and wherein the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, isadministered intramuscularly to a subject or subject population of 10 to18 years of age. In some embodiments, the subject or subject populationis from a dengue endemic region. According to some of these embodiments,the subject or subject population is seronegative for all four dengueserotypes.

In a preferred embodiment, the invention is directed a method ofpreventing dengue disease, and tetanus, diphtheria and pertussis in asubject comprising administering to the subject a subcutaneous injectionof the reconstituted unit dose of the invention as described herein andan intramuscular injection of the Tdap vaccine, in particular a combinedtetanus toxoid, reduced diphtheria toxoid and acellular pertussis(adsorbed) vaccine, such as BOOSTRIX®, according to the above describedadministration schedule, wherein the subject is between 10 years and 18years of age and from a dengue endemic region.

Unit Dose for Use in a Method of Prevention

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject, wherein the method also comprises the preventionof tetanus, diphtheria, and pertussis in the subject with a Tdapvaccine, in particular a combined tetanus toxoid, reduced diphtheriatoxoid and acellular pertussis (adsorbed) vaccine, such as BOOSTRIX®. Inparticular, the present invention is directed in part to a unit dose ofa dengue vaccine composition as described herein and a Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, for use in amethod of preventing dengue disease, and tetanus, diphtheria, andpertussis in a subject, respectively.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject population, wherein the method also comprises theprevention of tetanus, diphtheria, and pertussis in a subject populationwith a Tdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®. In particular, the present invention is directed in part to aunit dose of a dengue vaccine composition as described herein and a Tdapvaccine, in particular a combined tetanus toxoid, reduced diphtheriatoxoid and acellular pertussis (adsorbed) vaccine, such as BOOSTRIX®,for use in a method of preventing dengue disease, and tetanus,diphtheria, and pertussis in a subject population, respectively.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, for use in amethod of preventing dengue disease and tetanus, diphtheria, andpertussis in a subject population, comprising administering to thesubject population at least a first reconstituted unit dose of theinvention as described herein, wherein certain ratios of geometric meanneutralizing antibody titers (GMTs) at day 180 or 365 afteradministration of said first unit dose to the subject population areachieved, and concomitantly administering a Tdap vaccine, in particulara combined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, to the subjectpopulation. According to some embodiments, the geometric meanneutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and thegeometric mean neutralizing antibody titer for dengue serotype 4 (GMTDENV-4) when tested in at least 40, or at least 50, or at least 60subjects at day 180 or day 365 after at least a first administration ofsaid reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of GMT DENV-2:GMT DENV-4 of not more than 50, or notmore than 40, or not more than 30, or not more than 20. In some of theseembodiments, the ratio of GMT DENV-2:GMT DENV-1 is not more than 20, ornot more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof GMT DENV-2:GMT DENV-3 is not more than 20, or not more than 18, ornot more than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, for use in amethod of preventing dengue disease and tetanus, diphtheria, andpertussis in a subject comprising administering to the subject at leasta first reconstituted unit dose of the invention as described herein,wherein certain ratios of neutralizing antibody titers at day 180 or 365after administration of said first unit dose to the subject areachieved, and concomitantly administering a Tdap vaccine, in particulara combined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, to the subject.According to some embodiments, the neutralizing antibody titer fordengue serotype 2 and the neutralizing antibody titer for dengueserotype 4 at day 180 or day 365 after at least a first administrationof the reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of neutralizing antibody titer for DENV-2:neutralizingantibody titer for GMT DENV-4 of not more than 50, or not more than 40,or not more than 30, or not more than 20. In some of these embodiments,the ratio of the neutralizing antibody titers of DENV-2:DENV-1 is notmore than 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereinsaid unit dose and said Tdap vaccine are administered simultaneously. Insome of these embodiments, the simultaneous administration is on day 0or day 90, preferably on day 0. In other embodiments the administrationof said unit dose and said Tdap vaccine are done sequentially.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereinsaid unit dose is administered by subcutaneous injection and whereinsaid Tdap vaccine is administered by intramuscular injection. Accordingto some embodiments, the injections are administered to the arm,preferably to the deltoid region of the arm. According to some of theseembodiments, the subcutaneous injection of said unit dose and theintramuscular injection of said Tdap vaccine are administered todifferent anatomical sites, such as to opposite arms, in particular whenthe vaccines are administered simultaneously.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0/1 and at day 90. According to some ofthese embodiments a further third reconstituted unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12months after the first administration, such as 12 months (1 year) afterthe second administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereintwo reconstituted unit doses of the invention as described herein andone dose of the Tdap vaccine are administered, in particular accordingto the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and said Tdap vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to the unitdose of the invention as described herein and the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, for saiduses, wherein said unit dose is reconstituted and administeredsubcutaneously to a subject or subject population and said Tdap vaccineis administered intramuscularly to a subject or subject population, andwherein the subject or the subject population is seronegative withrespect to all dengue serotypes. In other embodiments, the subject orsubject population is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereinsaid unit dose and said Tdap vaccine are administered to a subject orsubject population from a dengue endemic region. In certain embodiments,the reconstituted unit dose of the invention as described herein isadministered subcutaneously and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, is administeredintramuscularly to a subject or subject population from a dengue endemicregion.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously and wherein the Tdap vaccine, in particulara combined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, is administeredintramuscularly to a subject or subject population of 10 to 18 years ofage. In some embodiments, the subject or subject population is from adengue endemic region. According to some of these embodiments, thesubject or subject population is seronegative for all four dengueserotypes.

In a preferred embodiment, the invention is directed to the unit dose ofthe invention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for use in a method ofpreventing dengue disease and tetanus, diphtheria and pertussis in asubject comprising administering to the subject a subcutaneous injectionof the reconstituted unit dose of the invention as described herein andan intramuscular injection of the Tdap vaccine, in particular a combinedtetanus toxoid, reduced diphtheria toxoid and acellular pertussis(adsorbed) vaccine, such as BOOSTRIX®, according to the above describedadministration schedule, wherein the subject is of 10 to 18 years of ageand from a dengue endemic region.

Use for the Manufacture of a Medicament in a Method of Prevention

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, further comprising the useof a Tdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, for the manufacture of a medicament for preventing tetanus,diphtheria, and pertussis in the subject. In particular, the presentinvention is directed in part to a use of a unit dose of a denguevaccine composition as described herein and a Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, for themanufacture of a medicament for preventing dengue disease and tetanus,diphtheria, and pertussis in a subject, respectively.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease, in a subject population, furthercomprising the use of a Tdap vaccine, in particular a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, for the manufacture of a medicament forpreventing tetanus, diphtheria, and pertussis in the subject population.In particular, the present invention is directed in part to a use of aunit dose of a dengue vaccination composition as described herein and aTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, for the manufacture of a medicament for preventing denguedisease and tetanus, diphtheria, and pertussis in a subject population,respectively.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for the manufacture ofa medicament for preventing dengue disease and tetanus, diphtheria, andpertussis, comprising administering to the subject population at least afirst reconstituted unit dose of the invention as described herein,wherein certain ratios of geometric mean neutralizing antibody titers(GMTs) at day 180 or 365 after administration of said first unit dose tothe subject population are achieved, and concomitantly administering aTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, to the subject population. According to some embodiments, thegeometric mean neutralizing antibody titer for dengue serotype 2 (GMTDENV-2) and the geometric mean neutralizing antibody titer for dengueserotype 4 (GMT DENV-4) when tested in at least 40, or at least 50, orat least 60 subjects at day 180 or day 365 after at least a firstadministration of said reconstituted unit dose of the invention asdescribed herein, and optionally a second administration of areconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, for the manufacture ofa medicament for preventing dengue disease and tetanus, diphtheria, andpertussis in a subject, comprising administering to the subject at leasta first reconstituted unit dose of the invention as described herein,wherein certain ratios of neutralizing antibody titers at day 180 or 365after administration of said first unit dose to the subject areachieved, and concomitantly administering a Tdap vaccine, in particulara combined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, to the subject.According to some embodiments, the neutralizing antibody titer fordengue serotype 2 and the neutralizing antibody titer for dengueserotype 4 at day 180 or day 365 after at least a first administrationof the reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of neutralizing antibody titer for DENV-2:neutralizingantibody titer for DENV-4 of not more than 50, or not more than 40, ornot more than 30, or not more than 20. In some of these embodiments, theratio of the neutralizing antibody titers of DENV-2:DENV-1 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the Tdap vaccine,in particular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, areadministered simultaneously. In some of these embodiments, thesimultaneous administration is on day 0 or day 90, preferably on day 0.In other embodiments, the administration of the unit dose of theinvention as described herein and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, are done sequentially.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein is reconstituted andadministered by subcutaneous injection and wherein the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, isadministered by intramuscular injection. According to some embodiments,the injections are administered to the arm, preferably to the deltoidregion of the arm. According to some of these embodiments thesubcutaneous injection of the unit dose of the invention as describedherein and the intramuscular injection of the Tdap vaccine, inparticular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, areadministered to different anatomical sites, such as to opposite arms, inparticular when the vaccines are administered simultaneously.

In certain embodiments, the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments, the two unit doses of the inventionas described herein are administered within 12 months or more, or withinsix months, or within three months, such as at day 0/1 and day 90.According to some of these embodiments a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments, the invention is directed to said uses, whereintwo unit doses of the invention as described herein and one dose of aTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, are administered, in particular according to the followingschedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and said Tdap vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.

In certain embodiments, the invention is directed to said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saiduses, wherein the unit dose of the invention as described herein isreconstituted and administered subcutaneously to a subject or subjectpopulation and the Tdap vaccine, in particular a combined tetanustoxoid, reduced diphtheria toxoid and acellular pertussis (adsorbed)vaccine, such as BOOSTRIX®, is administered intramuscularly to a subjector subject population, and wherein the subject or the subject populationis seronegative with respect to all dengue serotypes. In otherembodiments, the subject or subject population is seropositive withrespect to at least one dengue serotype.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the Tdap vaccine,in particular a combined tetanus toxoid, reduced diphtheria toxoid andacellular pertussis (adsorbed) vaccine, such as BOOSTRIX®, areadministered to a subject or subject population from a dengue endemicregion. In certain embodiments, the reconstituted unit dose of theinvention as described herein is administered subcutaneously and theTdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, is administered intramuscularly to a subject or subjectpopulation from a dengue endemic region.

In certain embodiments, the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously and the Tdap vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, is administeredintramuscularly to a subject or subject population of 10 to 18 years ofage. In some embodiments, the subject or subject population is from adengue endemic region. According to some of these embodiments, thesubject or subject population is seronegative for all four dengueserotypes.

In a preferred embodiment the invention is directed to the use of a unitdose of a dengue vaccine composition as described herein and the Tdapvaccine, in particular a combined tetanus toxoid, reduced diphtheriatoxoid and acellular pertussis (adsorbed) vaccine, such as BOOSTRIX®,for the manufacture of a medicament for preventing dengue disease andtetanus, diphtheria and pertussis in a subject comprising administeringto the subject a subcutaneous injection of the reconstituted unit doseof the invention as described herein and an intramuscular injection ofthe Tdap vaccine, in particular a combined tetanus toxoid, reduceddiphtheria toxoid and acellular pertussis (adsorbed) vaccine, such asBOOSTRIX®, according to the above described administration schedule,wherein the subject is of 10 to 18 years of age and from a dengueendemic region.

Method of Preventing Dengue Disease and Diphtheria, Tetanus, Pertussis,Poliomyelitis and Diseases Caused by Haemophilus influenzae Type b andUses Method of Prevention

The present invention is directed in part to a method of preventingdengue disease as well as diphtheria, tetanus, pertussis, poliomyelitisand diseases caused by Haemophilus influenzae type b in a subject. Thus,in certain embodiments the invention is directed to a method ofpreventing dengue disease in a subject, comprising administering to thesubject a reconstituted unit dose of the invention as described herein,wherein the method further comprises preventing diphtheria, tetanus,pertussis, poliomyelitis and diseases caused by Haemophilus influenzaetype b in the subject by concomitant administration of a DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, to the subject.

The present invention is directed in part to a method of preventingdengue disease as well as diphtheria, tetanus, pertussis, poliomyelitisand diseases caused by Haemophilus influenzae type b in a subjectpopulation. Thus, in certain embodiments the invention is directed to amethod of preventing dengue disease in a subject population, comprisingadministering to the subject population a reconstituted unit dose of theinvention as described herein, wherein the method further comprisespreventing diphtheria, tetanus, pertussis, poliomyelitis and diseasescaused by Haemophilus influenzae type b in the subject population byconcomitant administration of a DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®, to the subjectpopulation.

The present invention is in part directed to said method for preventingdengue disease and diphtheria, tetanus, pertussis, poliomyelitis anddiseases caused by Haemophilus influenzae type b in a subject populationcomprising administering to the subject population at least a firstreconstituted unit dose of the invention as described herein, whereincertain ratios of geometric mean neutralizing antibody titers (GMTs) atday 180 or 365 after administration of said first unit dose to thesubject population are achieved, and concomitantly administering aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, to the subject population. According to someembodiments, the geometric mean neutralizing antibody titer for dengueserotype 2 (GMT DENV-2) and the geometric mean neutralizing antibodytiter for dengue serotype 4 (GMT DENV-4) when tested in at least 40, orat least 50, or at least 60 subjects at day 180 or day 365 after atleast a first administration of said reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of GMT DENV-2:GMTDENV-4 of not more than 50, or not more than 40, or not more than 30, ornot more than 20. In some of these embodiments, the ratio of GMTDENV-2:GMT DENV-1 is not more than 20, or not more than 18, or not morethan 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of GMT DENV-2:GMT DENV-3 isnot more than 20, or not more than 18, or not more than 15 at day 180 or365 after administration of said first reconstituted unit dose.

The present invention is in part directed to said method for preventingdengue disease and diphtheria, tetanus, pertussis, poliomyelitis anddiseases caused by Haemophilus influenzae type b in a subject comprisingadministering to the subject at least a first reconstituted unit dose ofthe invention as described herein, wherein certain ratios ofneutralizing antibody titers at day 180 or 365 after administration ofsaid first unit dose to the subject are achieved, and concomitantlyadministering a DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, to the subject. According tosome embodiments, the neutralizing antibody titer for dengue serotype 2and the neutralizing antibody titer for dengue serotype 4 at day 180 orday 365 after at least a first administration of the reconstituted unitdose of the invention as described herein, and optionally a secondadministration of a reconstituted unit dose of the invention asdescribed herein 90 days after said first administration, provide aratio of neutralizing antibody titer for DENV-2:neutralizing antibodytiter for DENV-4 of not more than 50, or not more than 40, or not morethan 30, or not more than 20. In some of these embodiments, the ratio ofthe neutralizing antibody titers of DENV-2:DENV-1 is not more than 20,or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof the neutralizing antibody titers of DENV-2:DENV-3 is not more than20, or not more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said methods,wherein the unit dose of the invention as described herein and theDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, are administered simultaneously. In some of theseembodiments the simultaneous administration is on day 0 or day 90,preferably on day 0. In other embodiments the administration of the unitdose of the invention as described herein and the DTaP/IPV/Hib vaccine,in particular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, aredone sequentially.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered by subcutaneous injection and wherein the DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, is administered by intramuscular injection. According to someembodiments, the unit dose of the invention as described herein isadministered to the arm, preferably to the deltoid region of the arm.According to some embodiments, the subcutaneous injection of the unitdose of the invention as described herein and the intramuscularinjection of the DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, are administered to differentanatomical sites. According to some of these embodiments, said unit doseis administered subcutaneously to the arm, preferably to the deltoidregion, and said DTaP/IPV/Hib vaccine is administered intramuscularly tothe thigh, preferably to the anterolateral aspect of the thigh.

In certain embodiments the invention is directed to said methods,wherein two unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 month or more, or within 6month, or within three months, such as at day 0/1 and day 90. Accordingto some of these embodiments a further third unit dose of the inventionas described herein is administered after the second. Such a thirdadministration may act as a booster and may be administered between 6 to12 months after the first administration, such as 12 months after thefirst administration, or later than 12 month after the firstadministration, such as 12 months (1 year) after the secondadministration or even 5 years or longer after the first or secondadministration.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and four doses of a DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®, are administered, inparticular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said DTaP/IPV/Hib vaccine on day        0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first simultaneous administration, such as 2        months later and preferably on day 60,    -   a third administration of the second reconstituted unit dose        after said second administration, such as 3 months after the        first simultaneous administration and preferably on day 90,    -   a fourth administration of the third dose of said DTaP/IPV/Hib        vaccine after said third administration, such as 4 months after        the first simultaneous administration and preferably on day 120,        and    -   a fifth administration of the fourth dose of said DTaP/IPV/Hib        vaccine after said fourth administration, such as 9 to 12 months        later and preferably on day 390.

In certain embodiments the invention is directed to said methods,wherein two reconstituted unit doses of the invention as describedherein and four doses of a DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®, are administered, inparticular according to the following schedule

-   -   a first administration of the first dose of said DTaP/IPV/Hib        vaccine on day 0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first administration, such as 2 months later        and preferably on day 60,    -   a third administration of the third dose of said DTaP/IPV/Hib        vaccine after said second administration, such as 4 months after        the first administration and preferably on day 120,    -   a fourth simultaneous administration of the first reconstituted        unit dose and the fourth dose of said DTaP/IPV/Hib vaccine after        said third administration, such as 9 to 12 months later and        preferably on day 390, and    -   a fifth administration of the second reconstituted unit dose        after said fourth simultaneous administration, such as 3 months        later and preferably on day 480.

According to some embodiments, a fifth dose of a DTaP/IPV/Hib vaccine,in particular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, isadministered after the administration of the fourth dose of saidDTaP/IPV/Hib vaccine. For this purpose a commercially available combineddiphtheria, tetanus, pertussis, and poliomyelitis (DTaP-IPV) vaccinemarketed under the tradename Quadracel® from Sanofi Pasteur may be used.Quadracel® is a diphtheria and tetanus toxoids and acellular pertussisadsorbed and inactivated poliovirus vaccine and is indicated for activeimmunization against diphtheria, tetanus, pertussis and poliomyelitis. Asingle dose of Quadracel® is for use in children 4 through 6 years ofage as a fifth dose in the diphtheria, tetanus, pertussis vaccination(DTaP) series and as a fourth or fifth dose in the inactivatedpoliovirus vaccination (IPV) series in children who have received 4doses of Pentacel®. Accordingly, in some embodiments, a further sixthadministration of a fifth dose of a DTaP/IPV/Hib vaccine, in particulara combined DTaP/IPV/Hib vaccine, such as Quadracel®, may be conducted 30to 57 months after the administration of the fourth dose of aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®.

In certain embodiments, the invention is directed to said methods,wherein the subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saidmethods, wherein the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to a subject or subjectpopulation and the DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, is administered intramuscularlyto a subject or subject population, and wherein the subject or thesubject population is seronegative with respect to all dengue serotypes.In other embodiments, the subject or subject population is seropositivewith respect to at least one dengue serotype.

In certain embodiments, the invention is directed to said methods,wherein the unit dose of the invention as described herein and theDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, are administered to a subject or subject populationfrom a dengue endemic region. In certain embodiments, the reconstitutedunit dose of the invention as described herein is administeredsubcutaneously and the DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, is administered intramuscularlyto a subject or subject population from a dengue endemic region.

In certain embodiments the invention is directed to said methods,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously and wherein the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, isadministered intramuscularly to a subject or subject population of 2months to 4 years of age. In some embodiments the subject or subjectpopulation is from a dengue endemic region. In other embodiments, thesubject or subject population is from a dengue non-endemic region.According to some of these embodiments, the subject or subjectpopulation is seronegative for all four dengue serotypes.

In a preferred embodiment the invention is directed to a method ofpreventing dengue disease and diphtheria, tetanus, pertussis,poliomyelitis and diseases caused by Haemophilus influenzae type b in asubject comprising administering to the subject subcutaneous injectionsof the reconstituted unit dose of the invention as described herein andintramuscular injections of the DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®, according to one ofthe above described administration schedule, wherein the subject is asubject of 2 months to 4 years of age from a dengue endemic region.

Unit Dose for Use in a Method of Prevention

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject, wherein the method also comprises the preventionof diphtheria, tetanus, pertussis, poliomyelitis and diseases caused byHaemophilus influenzae type b in the subject with a DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®. In particular, the present invention is directed in part to aunit dose of a dengue vaccine composition as described herein and aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, for use in a method of preventing dengue disease anddiphtheria, tetanus, pertussis, poliomyelitis and diseases caused byHaemophilus influenzae type b in a subject, respectively.

The present invention is directed in part to the unit dose of theinvention as described herein for use in a method of preventing denguedisease in a subject population, wherein the method also comprises theprevention of diphtheria, tetanus, pertussis, poliomyelitis and diseasescaused by Haemophilus influenzae type b in the subject population with aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®. In particular, the present invention is directed inpart to a unit dose of a dengue vaccine composition as described hereinand a DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hibvaccine, such as Pentacel®, for use in a method of preventing denguedisease and diphtheria, tetanus, pertussis, poliomyelitis and diseasescaused by Haemophilus influenzae type b in a subject population,respectively.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for usein a method of preventing dengue disease and diphtheria, tetanus,pertussis, poliomyelitis and diseases caused by Haemophilus influenzaetype b in a subject population, comprising administering to the subjectpopulation at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of geometric mean neutralizingantibody titers (GMTs) at day 180 or 365 after administration of saidfirst unit dose to the subject population are achieved, andconcomitantly administering a DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®, to the subjectpopulation. According to some embodiments, the geometric meanneutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and thegeometric mean neutralizing antibody titer for dengue serotype 4 (GMTDENV-4) when tested in at least 40, or at least 50, or at least 60subjects at day 180 or day 365 after at least a first administration ofsaid reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of GMT DENV-2:GMT DENV-4 of not more than 50, or notmore than 40, or not more than 30, or not more than 20. In some of theseembodiments, the ratio of GMT DENV-2:GMT DENV-1 is not more than 20, ornot more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof GMT DENV-2:GMT DENV-3 is not more than 20, or not more than 18, ornot more than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

The present invention is in part directed to the unit dose of a denguevaccine composition as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for usein a method of preventing dengue disease and diphtheria, tetanus,pertussis, poliomyelitis and diseases caused by Haemophilus influenzaetype b in a subject comprising administering to the subject at least afirst reconstituted unit dose of the invention as described herein,wherein certain ratios of neutralizing antibody titers at day 180 or 365after administration of said first unit dose to the subject areachieved, and concomitantly administering a DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, to thesubject. According to some embodiments, the neutralizing antibody titerfor dengue serotype 2 and the neutralizing antibody titer for dengueserotype 4 at day 180 or day 365 after at least a first administrationof the reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of neutralizing antibody titer for DENV-2:neutralizingantibody titer for DENV-4 of not more than 50, or not more than 40, ornot more than 30, or not more than 20. In some of these embodiments, theratio of the neutralizing antibody titers of DENV-2:DENV-1 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose, and/or theratio of the neutralizing antibody titers of DENV-2:DENV-3 is not morethan 20, or not more than 18, or not more than 15 at day 180 or 365after administration of said first reconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein said unit dose and said DTaP/IPV/Hib vaccine areadministered simultaneously. In some of these embodiments thesimultaneous administration is on day 0 or day 90, preferably on day 0.In other embodiments the administration of said unit dose and saidDTaP/IPV/Hib vaccine are done sequentially.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein said unit dose is administered by subcutaneous injectionand wherein said DTaP/IPV/Hib vaccine is administered by intramuscularinjection. According to some embodiments, said unit dose is administeredto the arm, preferably to the deltoid region of the arm. According tosome embodiments, the subcutaneous injection of the unit dose of theinvention as described herein and the intramuscular injection of theDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, are administered to different anatomical sites.According to some of these embodiments, said unit dose is administeredsubcutaneously to the arm, preferably to the deltoid region, and saidDTaP/IPV/Hib vaccine is administered intramuscularly to the thigh,preferably to the anterolateral aspect of the thigh.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein two reconstituted unit doses of the invention as describedherein are administered. In some embodiments the two unit doses of theinvention as described herein are administered within 12 months or more,or within six months, or within three months, such as at day 0/1 and day90. According to some of these embodiments a further third reconstitutedunit dose of the invention as described herein is administered after thesecond administration. Such a third administration may act as a boosterand may be administered between 6 to 12 months after the firstadministration, such as 12 months after the first administration, orlater than 12 month after the first administration, such as 12 months (1year) after the second administration or even 5 years or longer afterthe first or second administration.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein two reconstituted unit doses of the invention as describedherein and four doses of the DTaP/IPV/Hib vaccine are administered, inparticular according to the following schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said DTaP/IPV/Hib vaccine on day        0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first simultaneous administration, such as 2        months later and preferably on day 60,    -   a third administration of the second reconstituted unit dose        after said second administration, such as 3 months after the        first simultaneous administration and preferably on day 90,    -   a fourth administration of the third dose of said DTaP/IPV/Hib        vaccine after said third administration, such as 4 months after        the first simultaneous administration and preferably on day 120,        and    -   a fifth administration of the fourth dose of said DTaP/IPV/Hib        vaccine after said fourth administration, such as 9 to 12 months        later and preferably on day 390.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein two reconstituted unit doses of the invention as describedherein and four doses of the DTaP/IPV/Hib vaccine are administered, inparticular according to the following schedule

-   -   a first administration of the first dose of said DTaP/IPV/Hib        vaccine on day 0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first administration, such as 2 months later        and preferably on day 60,    -   a third administration of the third dose of said DTaP/IPV/Hib        vaccine after said second administration, such as 4 months after        the first administration and preferably on day 120,    -   a fourth simultaneous administration of the first reconstituted        unit dose and the fourth dose of said DTaP/IPV/Hib vaccine after        said third administration, such as 9 to 12 months later and        preferably on day 390, and    -   a fifth administration of the second reconstituted unit dose        after said fourth simultaneous administration, such as 3 months        later and preferably on day 480.

According to some embodiments, a fifth dose of a DTaP/IPV/Hib vaccine,in particular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, isadministered after the administration of the fourth dose of saidDTaP/IPV/Hib vaccine. For this purpose a commercially available combineddiphtheria, tetanus, pertussis, and poliomyelitis (DTaP-IPV) vaccinemarketed under the tradename Quadracel® from Sanofi Pasteur may be used.Quadracel® is a diphtheria and tetanus toxoids and acellular pertussisadsorbed and inactivated poliovirus vaccine and is indicated for activeimmunization against diphtheria, tetanus, pertussis and poliomyelitis. Asingle dose of Quadracel® is for use in children 4 through 6 years ofage as a fifth dose in the diphtheria, tetanus, pertussis vaccination(DTaP) series and as a fourth or fifth dose in the inactivatedpoliovirus vaccination (IPV) series in children who have received 4doses of Pentacel®. Accordingly, in some embodiments, a further sixthadministration of a fifth dose of a DTaP/IPV/Hib vaccine, in particulara combined DTaP/IPV/Hib vaccine, such as Quadracel®, may be conducted 30to 57 months after the administration of the fourth dose of aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein the subject or subject population is seronegative to alldengue serotypes. In certain embodiments, the invention is directed tothe unit dose of the invention as described herein and the DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, for said uses, wherein said unit dose is reconstituted andadministered subcutaneously to a subject or subject population and saidDTaP/IPV/Hib vaccine is administered intramuscularly to a subject orsubject population, and wherein the subject or the subject population isseronegative with respect to all dengue serotypes. In other embodiments,the subject or subject population is seropositive with respect to atleast one dengue serotype.

In certain embodiments, the invention is directed to the unit dose ofthe invention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein said unit dose and said DTaP/IPV/Hib vaccine areadministered to a subject or subject population from a dengue endemicregion. In certain embodiments, the reconstituted unit dose of theinvention as described herein is administered subcutaneously and theDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, is administered intramuscularly to a subject orsubject population from a dengue endemic region.

In certain embodiments the invention is directed to the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for saiduses, wherein the reconstituted unit dose of the invention as describedherein is administered subcutaneously and wherein the DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, is administered intramuscular to a subject or subjectpopulation of 2 months to 4 years of age. In some embodiments thesubject or subject population is from a dengue endemic region. In otherembodiments, the subject or subject population is from a denguenon-endemic region. According to some of these embodiments, the subjector subject population is seronegative for all four dengue serotypes.

In a preferred embodiment the invention is directed to the unit dose ofthe invention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for usein a method of preventing dengue disease and diphtheria, tetanus,pertussis, poliomyelitis and diseases caused by Haemophilus influenzaetype b in a subject comprising administering to the subject subcutaneousinjections of the reconstituted unit dose of the invention as describedherein and intramuscular injections of the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, accordingto one of the above described administration schedule, wherein thesubject is a subject of 2 months to 4 years of age from a dengue endemicregion.

Use for the Manufacture of a Medicament in a Method of Prevention

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject, further comprising the useof a DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hibvaccine, such as Pentacel®, for the manufacture of a medicament forpreventing diphtheria, tetanus, pertussis, poliomyelitis and diseasescaused by Haemophilus influenzae type b in the subject. In particular,the present invention is directed in part to a use of a unit dose of adengue vaccine composition as described herein and a DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, for the manufacture of a medicament for preventing denguedisease and diphtheria, tetanus, pertussis, poliomyelitis and diseasescaused by Haemophilus influenzae type b in a subject, respectively.

The present invention is directed in part to the use of a unit dose ofthe invention as described herein for the manufacture of a medicamentfor preventing dengue disease in a subject population, furthercomprising the use of a DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, for the manufacture of amedicament for preventing diphtheria, tetanus, pertussis, poliomyelitisand diseases caused by Haemophilus influenzae type b in the subjectpopulation. In particular, the present invention is directed in part toa use of a unit dose of a dengue vaccine composition as described hereinand a DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hibvaccine, such as Pentacel®, for the manufacture of a medicament forpreventing dengue disease and diphtheria, tetanus, pertussis,poliomyelitis and diseases caused by Haemophilus influenzae type b in asubject population, respectively.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for themanufacture of a medicament for preventing dengue disease anddiphtheria, tetanus, pertussis, poliomyelitis and diseases caused byHaemophilus influenzae type b in a subject population, comprisingadministering to the subject population at least a first reconstitutedunit dose of the invention as described herein, wherein certain ratiosof geometric mean neutralizing antibody titers (GMTs) at day 180 or 365after administration of said first unit dose to the subject populationare achieved, and concomitantly administering a DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, to thesubject population. According to some embodiments, the geometric meanneutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and thegeometric mean neutralizing antibody titer for dengue serotype 4 (GMTDENV-4) when tested in at least 40, or at least 50, or at least 60subjects at day 180 or day 365 after at least a first administration ofsaid reconstituted unit dose of the invention as described herein, andoptionally a second administration of a reconstituted unit dose of theinvention as described herein 90 days after said first administration,provide a ratio of GMT DENV-2:GMT DENV-4 of not more than 50, or notmore than 40, or not more than 30, or not more than 20. In some of theseembodiments, the ratio of GMT DENV-2:GMT DENV-1 is not more than 20, ornot more than 18, or not more than 15 at day 180 or 365 afteradministration of said first reconstituted unit dose, and/or the ratioof GMT DENV-2:GMT DENV-3 is not more than 20, or not more than 18, ornot more than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

The present invention is in part directed to the use of a unit dose ofthe invention as described herein and a DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, for themanufacture of a medicament for preventing dengue disease anddiphtheria, tetanus, pertussis, poliomyelitis and diseases caused byHaemophilus influenzae type b in a subject, comprising administering tothe subject at least a first reconstituted unit dose of the invention asdescribed herein, wherein certain ratios of neutralizing antibody titersat day 180 or 365 after administration of said first unit dose to thesubject are achieved, and concomitantly administering a DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, to the subject. According to some embodiments, theneutralizing antibody titer for dengue serotype 2 and the neutralizingantibody titer for dengue serotype 4 at day 180 or day 365 after atleast a first administration of the reconstituted unit dose of theinvention as described herein, and optionally a second administration ofa reconstituted unit dose of the invention as described herein 90 daysafter said first administration, provide a ratio of neutralizingantibody titer for DENV-2:neutralizing antibody titer for DENV-4 of notmore than 50, or not more than 40, or not more than 30, or not more than20. In some of these embodiments, the ratio of the neutralizing antibodytiters of DENV-2:DENV-1 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose, and/or the ratio of the neutralizing antibodytiters of DENV-2:DENV-3 is not more than 20, or not more than 18, or notmore than 15 at day 180 or 365 after administration of said firstreconstituted unit dose.

In some embodiments, the geometric mean neutralizing antibody titers(GMTs) of a subject population or the neutralizing antibody titers of asubject are determined in accordance with a microneutralization test,for example according to the method described in Example 2.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, are administered simultaneously. In some of these embodimentsthe simultaneous administration is on day 0 or day 90, preferably on day0. In other embodiments the administration of the unit dose of theinvention as described herein and the DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, are donesequentially.

In certain embodiments the invention is directed to said uses, whereinthe unit dose of the invention as described herein is reconstituted andadministered by subcutaneous injection and wherein the DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, is administered by intramuscular injection. According to someembodiments, the unit dose of the invention as described herein isadministered to the arm, preferably to the deltoid region of the arm.According to some embodiments, the subcutaneous injection of the unitdose of the invention as described herein and the intramuscularinjection of the DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, are administered to differentanatomical sites. According to some of these embodiments, said unit doseis administered subcutaneously to the arm, preferably to the deltoidregion, and said DTaP/IPV/Hib vaccine is administered intramuscularly tothe thigh, preferably to the anterolateral aspect of the thigh.

In certain embodiments the invention is directed to said uses, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments the two unit doses of the invention asdescribed herein are administered within 12 months or more, or withinsix months, or within three months, such as at day 0/1 and day 90.According to some of these embodiments a further third unit dose of theinvention as described herein is administered after the secondadministration. Such a third administration may act as a booster and maybe administered between 6 to 12 months after the first administration,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and four doses of aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, are administered, in particular according to thefollowing schedule

-   -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said DTaP/IPV/Hib vaccine on day        0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first simultaneous administration, such as 2        months later and preferably on day 60,    -   a third administration of the second reconstituted unit dose        after said second administration, such as 3 months after the        first simultaneous administration and preferably on day 90,    -   a fourth administration of the third dose of said DTaP/IPV/Hib        vaccine after said third administration, such as 4 months after        the first simultaneous administration and preferably on day 120,        and    -   a fifth administration of the fourth dose of said DTaP/IPV/Hib        vaccine after said fourth administration, such as 9 to 12 months        later and preferably on day 390.

In certain embodiments the invention is directed to said uses, whereintwo unit doses of the invention as described herein and four doses of aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, are administered, in particular according to thefollowing schedule

-   -   a first administration of the first dose of said DTaP/IPV/Hib        vaccine on day 0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first administration, such as 2 months later        and preferably on day 60,    -   a third administration of the third dose of said DTaP/IPV/Hib        vaccine after said second administration, such as 4 months after        the first administration and preferably on day 120,    -   a fourth simultaneous administration of the first reconstituted        unit dose and the fourth dose of said DTaP/IPV/Hib vaccine after        said third administration, such as 9 to 12 months later and        preferably on day 390, and    -   a fifth administration of the second reconstituted unit dose        after said fourth simultaneous administration, such as 3 months        later and preferably on day 480.

According to some embodiments, a fifth dose of a DTaP/IPV/Hib vaccine,in particular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, isadministered after the administration of the fourth dose of saidDTaP/IPV/Hib vaccine. For this purpose a commercially available combineddiphtheria, tetanus, pertussis, and poliomyelitis (DTaP-IPV) vaccinemarketed under the tradename Quadracel® from Sanofi Pasteur may be used.Quadracel® is a diphtheria and tetanus toxoids and acellular pertussisadsorbed and inactivated poliovirus vaccine and is indicated for activeimmunization against diphtheria, tetanus, pertussis and poliomyelitis. Asingle dose of Quadracel® is for use in children 4 through 6 years ofage as a fifth dose in the diphtheria, tetanus, pertussis vaccination(DTaP) series and as a fourth or fifth dose in the inactivatedpoliovirus vaccination (IPV) series in children who have received 4doses of Pentacel®. Accordingly, in some embodiments, a further sixthadministration of a fifth dose of a DTaP/IPV/Hib vaccine, in particulara combined DTaP/IPV/Hib vaccine, such as Quadracel®, may be conducted 30to 57 months after the administration of the fourth dose of aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®.

In certain embodiments, the invention is directed to said uses, whereinthe subject or subject population is seronegative to all dengueserotypes. In certain embodiments, the invention is directed to saiduses, wherein the unit dose of the invention as described herein isreconstituted and administered subcutaneously to a subject or subjectpopulation and the DTaP/IPV/Hib vaccine, in particular a combinedDTaP/IPV/Hib vaccine, such as Pentacel®, is administered intramuscularlyto a subject or subject population, and wherein the subject or thesubject population is seronegative with respect to all dengue serotypes.In other embodiments, the subject or subject population is seropositivewith respect to at least one dengue serotype.

In certain embodiments, the invention is directed to said uses, whereinthe unit dose of the invention as described herein and the DTaP/IPV/Hibvaccine, in particular a combined DTaP/IPV/Hib vaccine, such asPentacel®, are administered to a subject or subject population from adengue endemic region. In certain embodiments, the reconstituted unitdose of the invention as described herein is administered subcutaneouslyand the DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hibvaccine, such as Pentacel®, is administered intramuscularly to a subjector subject population from a dengue endemic region.

In certain embodiments the invention is directed to said uses, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously and the DTaP/IPV/Hib vaccine, in particulara combined DTaP/IPV/Hib vaccine, such as Pentacel®, is administeredintramuscularly to a subject or subject population of 2 months to 4years of age. In some embodiments the subject or subject population isfrom a dengue endemic region. In other embodiments, the subject orsubject population is from a dengue non-endemic region. According tosome of these embodiments, the subject or subject population isseronegative for all four dengue serotypes.

In a preferred embodiment the invention is directed to the use of a unitdose of a dengue vaccine composition as described herein and aDTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib vaccine,such as Pentacel®, for the manufacture of a medicament for preventingdengue disease and diphtheria, tetanus, pertussis, poliomyelitis anddiseases caused by Haemophilus influenzae type bin a subject comprisingadministering to the subject subcutaneous injections of thereconstituted unit dose of the invention as described herein andintramuscular injections of the DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®, according to one ofthe above described administration schedule, wherein the subject is asubject of 2 months to 4 years of age from a dengue endemic region.

Method of Prevention Using Multiple Co-Vaccinations

In case multiple disease should be prevented the unit dose according tothe invention (TDV) can be co-administered in at least any of thefollowing combinations.

Double Vaccine Combinations: TDV+YF, TDV+Hepatitis A, TDV+MMR, TDV+Tdap,TDV+DTap, or TDV+HPV Triple Vaccine Combinations:

TDV+YF+Hepatitis A, TDV+YF+MMR, TDV+YF+Tdap, TDV+YF+Dtap, TDV+YF+HPV,TDV+Hepatitis A+MMR, TDV+Hepatitis A+Tdap, TDV+Hepatitis A+DTap,TDV+Hepatitis A+HPV, TDV+MMR+Tdap, TDV+MMR+DTap, TDV+MMR+HPV,TDV+Tdap+DTap, TDV+Tdap+HPV, or TDV+DTap+HPV.

Quadruple Vaccine Combinations

TDV+YF+Hepatitis A+MMR, TDV+YF+Hepatitis A+Tdap, TDV+YF+HepatitisA+DTap, TDV+YF+Hepatitis A+HPV, TDV+YF+MMR+Tdap, TDV+YF+MMR+DTap,TDV+YF+MMR+HPV, TDV+YF+Tdap+DTap, TDV+YF+Tdap+HPV, TDV+YF+DTap+HPV,TDV+Hepatitis A+MMR+Tdap, TDV+Hepatitis A+MMR+DTap, TDV+HepatitisA+MMR+HPV, TDV+Hepatitis A+Tdap+DTap, TDV+Hepatitis A+Tdap+HPV,TDV+Hepatitis A+DTap+HPV, TDV+MMR+Tdap+DTap, TDV+MMR+Tdap+HPV,TDV+MMR+DTap+HPV, or TDV+Tdap+DTap+HPV.

Quintuple Vaccine Combinations

TDV+YF+Hepatitis A+MMR+Tdap, TDV+YF+Hepatitis A+MMR+Dtap,TDV+YF+Hepatitis A+MMR+HPV, TDV+YF+Hepatitis A+Tdap+DTap,TDV+YF+Hepatitis A+Tdap+HPV, TDV+YF+Hepatitis A+DTap+HPV,TDV+YF+MMR+Tdap+DTap, TDV+YF+MMR+Tdap+HPV, TDV+YF+MMR+DTap+HPV,TDV+YF+Tdap+DTap+HPV, TDV+Hepatitis A+MMR+Tdap+DTap, TDV+HepatitisA+MMR+Tdap+HPV, TDV+Hepatitis A+MMR+DTap+HPV, TDV+HepatitisA+Tdap+Dtap+HPV, or TDV+MMR+Tdap+DTap+HPV.

Sextuple Vaccine Combinations

TDV+YF+Hepatitis A+MMR+Tdap+DTap, TDV+YF+Hepatitis A+MMR+Tdap+HPV,TDV+YF+Hepatitis A+MMR+DTap+HPV, TDV+YF+Hepatitis A+Tdap+DTap+HPV,TDV+YF+MMR+Tdap+DTap+HPV, or TDV+Hepatitis A+MMR+Tdap+DTap+HPV.

Septuple Vaccine Combinations TDV+YF+Hepatitis A+MMR+Tdap+DTap+HPV.

A preferred combination is e.g. TDV, YF and MMR.

Method of Co-Vaccinations with Other Dengue Vaccines

In certain embodiments of the invention the method is directed to aco-administration with other dengue vaccines such as Dengvaxia®.Dengvaxia® is a tetravalent dengue vaccine based on a yellow feverbackbone, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), and hasbeen licensed in several countries based on the clinical demonstrationof an overall vaccine efficacy (VE) against virologically-confirmeddengue (VCD) of 56-61% in children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent denguevaccine in healthy children in Asia: a phase 3, randomised,observer-masked, placebo-controlled trial. Lancet 2014, 384:1358-65;Villar L A et al. Safety and immunogenicity of a recombinant tetravalentdengue vaccine in 9-16 year olds: a randomized, controlled, phase IItrial in Latin America. Pediatr Infect Dis J 2013, 32:1102-9). Thepreparation of these particular strains CYD1, CYD2, CYD3 and CYD4 hasbeen described in detail in international patent applications WO98/37911, WO 03/101397, WO07/021672, WO 08/007021, WO 08/047023 and WO08/065315, to which reference may be made for a precise description ofthe processes for their preparation. The corresponding nucleotidesequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are providedin WO2016034629 and SEQ ID NOs are set out in Table 16.

Method of Co-Vaccinations with Other Travel Vaccines

In certain embodiments of the invention the method is directed to aco-administration with other travel vaccines such as Cholera, HepatitisA, Hepatitis E, Japanese encephalitis, Meningococcal disease, Rabies,Tick-borne encephalitis, Typhoid fever, and Yellow fever.

According to one embodiment the dengue vaccine according to theinvention is co-administered with all of the above vaccines.

According to one embodiment the dengue vaccine according to theinvention is co-administered with one ore more of the above vaccines.

Method of Stimulating an Immune Response and Uses Method of Stimulatingan Immune Response

In certain embodiments the invention is directed to a method forstimulating an immune response, preferably a balanced immune response,to all four dengue serotypes in a subject, comprising administering tothe subject a reconstituted unit dose of the invention as describedherein.

In certain embodiments, the method includes a reconstituted unitdose/tetravalent dengue virus composition of a dengue vaccinecomposition administered for stimulating an immune response, preferablya balanced immune response, to all four dengue serotypes in a subject orsubject population, the reconstituted unit dose comprising: atetravalent virus composition including four live attenuated denguevirus strains, wherein the unit dose, described herein, is lyophilizedand upon reconstitution with 0.5 mL of a pharmaceutically acceptablediluent the reconstituted unit dose is obtained, wherein thereconstituted unit dose comprises:

(i) a dengue serotype 1, such as a chimeric dengue serotype 2/1 strain,in a concentration of at least 3.3 log 10 pfu/0.5 ml,(ii) a dengue serotype 2, such as a dengue serotype 2 strain, in aconcentration of at least 2.7 log 10 pfu/0.5 ml,(iii) a dengue serotype 3, such as a chimeric dengue serotype 2/3strain, in a concentration of at least 4.0 log 10 pfu/0.5 ml, and(vi) a dengue serotype 4, such as a chimeric dengue serotype 2/4 strain,in a concentration of at least 4.5 log 10 pfu/0.5 ml.

It is preferred that the reconstituted unit dose/tetravalent denguevirus composition is used in the method of stimulating an immuneresponse of the present invention, wherein upon reconstitution of theunit dose with a pharmaceutically acceptable diluent dengue serotypes(i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mLand based on said total concentration the concentration of (ii) inpfu/0.5 mL is less than 2%, the concentration of (iv) in pfu/0.5 mL isat least 50%, the concentration of (i) in pfu/0.5 mL is at least 1%, andthe concentration of (iii) in pfu/0.5 mL is at least 6% and wherein thesubject or subject population is of 18 to 60 years of age.

In another preferred embodiment, the reconstituted unit dose/tetravalentdengue virus composition is used in the method of stimulating an immuneresponse of the present invention, wherein upon reconstitution with apharmaceutically acceptable diluent dengue serotypes (i), (ii), (iii),and (iv) provide a total concentration of pfu/0.5 mL and based on saidtotal concentration the concentration of (ii) in pfu/0.5 mL is less than10%, and the concentration of (iv) in pfu/0.5 mL is at least 50%, andthe concentration of (i) in pfu/0.5 mL is at least 1%, and theconcentration of (iii) in pfu/0.5 mL is at least 8% and wherein thesubject or subject population is of 2 to 17 years of age.

The present invention is in part directed to a method for stimulating animmune response to all four serotypes of dengue virus in a subject,comprising administering to the subject a reconstituted unit dose of theinvention as described herein by subcutaneous injection.

In certain embodiments, the invention is directed to said method,wherein the immune response to all four serotypes of dengue virus isbalanced.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose is administered by subcutaneousinjection to the arm, preferably to the deltoid region of the arm.

In certain embodiments, the invention is directed to said method,wherein the subject is seronegative to all dengue serotypes.

In certain embodiments, the invention is directed to said method,wherein two reconstituted unit doses of the invention as describedherein are administered. In some embodiments, the two reconstituteddoses are administered within 12 months or more, or within six months,or within three months, such as at day 0 and day 90 or at day 1 and day90. According to some of these embodiments, a third unit dose of theinvention as described herein is administered between 6 and 12 monthsafter the administration of said first unit dose, such as 12 monthsafter the first administration, or later than 12 month after the firstadministration, such as 12 months after the second administration.

In certain embodiments, the invention is directed to said method,wherein the unit dose of the invention as described herein isadministered to a subject from a dengue endemic region. In some of theseembodiments, the subject is from Singapore, Dominican Republic, Panama,Philippines, Colombia, Puerto Rico or Thailand, in particular fromSingapore, Dominican Republic, Panama, or Philippines. In otherembodiments, the subject is from a dengue non-endemic region. Such asubject may be subject to a vaccination according to the invention inthe context of traveling to a dengue endemic region. In certainembodiments, the reconstituted unit dose of the invention as describedherein is administered subcutaneously to a subject that is from a dengueendemic region or a dengue non-endemic region.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously to a subject that is seronegative withrespect to all dengue serotypes. In other embodiments, the subject isseropositive with respect to at least one dengue serotype.

In certain embodiments, the invention is directed to said method,wherein the neutralizing antibody titers of the subject when tested atday 180 or day 365 after at least a first administration of said unitdose, and optionally a second administration of said unit dose 90 daysafter said first administration, provide a ratio of not more than 50, ornot more than 40, or nor more than 30, or not more than 20 for theneutralizing antibody titer of dengue serotype 2 to the neutralizingantibody titer of dengue serotype 4. In certain embodiments, saidneutralizing antibody titers of the subject further provide a ratio ofnot more than 20 for the neutralizing antibody titer of dengue serotype2 to the neutralizing antibody titer of dengue serotype 1, and/or aratio of not more than 20 for the neutralizing antibody titer of dengueserotype 2 to the neutralizing antibody titer of dengue serotype 3.

In certain embodiments, the invention is directed to said method,wherein the unit dose of the invention as described herein isadministered to a subject of 2 to 60 years of age, or more than 17years, or more than 18 years, or 18 to 60 years of age. In certainembodiments the subject is 1 to 17 years of age, or less than 9 years ofage, or less than 4 years of age or less than 2 years of age. In furtherembodiments, the subjects or subject population are adults of more than21 years, or 21 to 60 years, or 21 to 45 years of age. According to someof these embodiments the subject is seronegative and from adengue-endemic region.

In certain embodiments, the invention is directed to said method,wherein the unit dose of the invention as described herein isadministered to a pediatric subject of less than 2 years of age,preferably of 2 months to 2 years of age or 2 months to 1.5 years of ageor 2 months to 1 year of age. According to some of these embodiments,the pediatric subject is seronegative and from a dengue endemic region.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously to a pediatric subject of less than 2years of age, preferably of 2 months to 2 years of age or 2 months to1.5 years of age or 2 months to 1 year of age. According to some ofthese embodiments, the pediatric subject is seronegative and from adengue endemic region.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered concomitantly with another vaccine. In one embodiments,the reconstituted unit dose of the invention as described herein isadministered concomitantly with a yellow fever vaccine, in particularYF-17D. It is particularly preferred that the reconstituted unit dose ofthe invention as described herein is administered concomitantly with ayellow fever vaccine, in particular YF-17D, as described in the previoussection.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered concomitantly with another vaccine. In one embodiments,the reconstituted unit dose of the invention as described herein isadministered concomitantly with a HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9. It is particularly preferred that thereconstituted unit dose of the invention as described herein isadministered concomitantly with a HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, as described in the previous section.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered concomitantly with another vaccine. In one embodiments,the reconstituted unit dose of the invention as described herein isadministered concomitantly with a MMR vaccine, such as M-M-R® II. It isparticularly preferred that the reconstituted unit dose of the inventionas described herein is administered concomitantly with a MMR vaccine,such as M-M-R® II, as described in the previous section.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered concomitantly with another vaccine. In one embodiments,the reconstituted unit dose of the invention as described herein isadministered concomitantly with a DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®. It is particularlypreferred that the reconstituted unit dose of the invention as describedherein is administered concomitantly with a DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, asdescribed in the previous section.

In certain embodiments the invention is directed to a method forstimulating an immune response, preferably a balanced immune response,to all four dengue serotypes in an elderly subject, comprisingadministering to the elderly subject a reconstituted unit dose of theinvention as described herein.

The present invention is in part directed to a method for stimulating animmune response to all four serotypes of dengue virus in an elderlysubject, comprising administering to the elderly subject a reconstitutedunit dose of the invention as described herein by subcutaneousinjection.

In certain embodiments, the invention is directed to said method,wherein the immune response to all four serotypes of dengue virus isbalanced.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose is administered by subcutaneousinjection to the arm, preferably to the deltoid region of the arm.

In certain embodiments, the invention is directed to said method,wherein the elderly subject is seronegative to all dengue serotypes.

In certain embodiments, the invention is directed to said method,wherein two reconstituted unit doses of the invention as describedherein are administered. In some embodiments, the two reconstituteddoses are administered within 12 months or more, or within six months,or within three months, such as at day 0 and day 90. According to someof these embodiments, a third unit dose of the invention as describedherein is administered between 6 and 12 months after the administrationof said first unit dose, such as 12 months after the firstadministration, or later than 12 month after the first administration,such as 12 months after the second administration.

In certain embodiments, the invention is directed to said method,wherein the unit dose of the invention as described herein isadministered to an elderly subject from a dengue endemic region. In someof these embodiments, the elderly subject is from Singapore, DominicanRepublic, Panama, Philippines, Colombia, Puerto Rico or Thailand, inparticular from Singapore, Dominican Republic, Panama, or Philippines.In other embodiments, the elderly subject is from a dengue non-endemicregion. Such an elderly subject may be subject to a vaccinationaccording to the invention in the context of traveling to a dengueendemic region. In certain embodiments, the reconstituted unit dose ofthe invention as described herein is administered subcutaneously to anelderly subject that is from a dengue endemic region or a denguenon-endemic region.

In certain embodiments, the invention is directed to said method,wherein the reconstituted unit dose of the invention as described hereinis administered subcutaneously to an elderly subject that isseronegative with respect to all dengue serotypes. In other embodiments,the elderly subject is seropositive with respect to at least one dengueserotype.

In certain embodiments, the invention is directed to said method,wherein the elderly subject has at least one chronic condition ordisease. The at least one chronic condition or disease may be selectedfrom diabetes, hypertension, allergies, previous strokes, ischemic heartdisease, chronic renal impairment and chronic obstructive pulmonarydisease.

In certain embodiments, the invention is directed to said method,wherein the elderly subject has an impaired immune system.

Use for the Manufacture of a Medicament for Stimulating an ImmuneResponse

The present invention is in part directed to the use of thereconstituted unit dose of the invention as described herein for themanufacture of a medicament for stimulating an immune response to allfour serotypes of dengue virus in a subject. In one embodiment areconstituted unit dose of the invention as described herein isadministered by subcutaneous injection.

In certain embodiments, the present invention is directed to a use ofthe reconstituted unit dose/tetravalent dengue virus composition of adengue vaccine composition for the manufacture of a medicament forstimulating an immune response to all four serotypes of dengue virus ina subject or in a subject population, wherein the reconstituted unitdose comprises a tetravalent virus composition including four liveattenuated dengue virus strains, wherein a unit dose, as describedherein, is lyophilized and upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent the reconstituted unit dose isobtained, wherein the reconstituted unit dose comprises:

(i) a dengue serotype 1, such as a chimeric dengue serotype 2/1 strain,in a concentration of at least 3.3 log 10 pfu/0.5 ml,(ii) a dengue serotype 2, such as a dengue serotype 2 strain, in aconcentration of at least 2.7 log 10 pfu/0.5 ml,(iii) a dengue serotype 3, such as a chimeric dengue serotype 2/3strain, in a concentration of at least 4.0 log 10 pfu/0.5 ml, and(vi) a dengue serotype 4, such as a chimeric dengue serotype 2/4 strain,in a concentration of at least 4.5 log 10 pfu/0.5 ml.

It is preferred that the use of the reconstituted unit dose/tetravalentdengue virus composition for the manufacture of a medicament in a methodfor stimulating an immune response to all four serotypes of dengue virusin a subject or in a subject population is obtained upon reconstitutionof the unit dose with a pharmaceutically acceptable diluent and thedengue serotypes (i), (ii), (iii), and (iv) provide a totalconcentration of pfu/0.5 mL and based on said total concentration theconcentration of (ii) in pfu/0.5 mL is less than 2%, the concentrationof (iv) in pfu/0.5 mL is at least 50%, the concentration of (i) inpfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mLis at least 6% and wherein the subject or subject population is of 18 to60 years of age.

In another preferred embodiment, the use of reconstituted unitdose/tetravalent dengue virus composition for the manufacture of amedicament for a method for stimulating an immune response to all fourserotypes of dengue virus in a subject or in a subject population isobtained upon reconstitution of the unit dose described herein with apharmaceutically acceptable diluent and the dengue serotypes (i), (ii),(iii), and (iv) provide a total concentration of pfu/0.5 mL and based onsaid total concentration the concentration of (ii) in pfu/0.5 mL is lessthan 10%, and the concentration of (iv) in pfu/0.5 mL is at least 50%,and the concentration of (i) in pfu/0.5 mL is at least 1%, and theconcentration of (iii) in pfu/0.5 mL is at least 8% and wherein thesubject or subject population is of 2 to 17 years of age.

In certain embodiments, the invention is directed to said use, whereinthe immune response to all four serotypes of dengue virus is balanced.

In certain embodiments, the invention is directed to said use, whereinthe reconstituted unit dose is administered by subcutaneous injection tothe arm, preferably to the deltoid region of the arm.

In certain embodiments, the invention is directed the reconstituted unitdose of the invention as described herein for said use, wherein thesubject is seronegative to all dengue serotypes.

In certain embodiments, the invention is directed to said use, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments, the two reconstituted unit doses areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0 and day 90 or at day 1 and day 90.According to some of these embodiments, a third reconstituted unit doseis administered 6 to 12 months after the administration of the firstreconstituted unit dose, such as 12 months after the firstadministration, or later than 12 month after the first administration,such as 12 months (1 year) after the second administration or even 5years or longer after the first or second administration.

In certain embodiments, the invention is directed to said use, whereinthe subject is from a dengue endemic region. In other embodiments, thesubject is from a dengue non-endemic region. Such a subject may besubject to a vaccination according to the invention in the context oftraveling to a dengue endemic region. In certain embodiments, thereconstituted unit dose of the invention as described herein isadministered subcutaneously to a subject that is from a dengue endemicregion or a dengue non-endemic region.

In certain embodiments, the invention is directed to said use, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously to a subject that is seronegative withrespect to all dengue serotypes. In other embodiments, the subject isseropositive with respect to at least one dengue serotype.

In certain embodiments, the invention is directed to said use, whereinthe neutralizing antibody titers of the subject when tested at day 180or day 365 after at least a first administration of said unit dose, andoptionally a second administration of said unit dose 90 days after saidfirst administration, provide a ratio of not more than 50, or not morethan 40, or nor more than 30, or not more than 20 for the neutralizingantibody titer of dengue serotype 2 to the neutralizing antibody titerof dengue serotype 4. In certain embodiments, said neutralizing antibodytiters of the subject further provide a ratio of not more than 20 forthe neutralizing antibody titer of dengue serotype 2 to the neutralizingantibody titer of dengue serotype 1, and/or a ratio of not more than 20for the neutralizing antibody titer of dengue serotype 2 to theneutralizing antibody titer of dengue serotype 3.

In certain embodiments, the invention is directed to said use, whereinthe reconstituted unit dose of the invention as described herein isadministered to a subject of 2 to 60 years of age or more than 17 years,or more than 18 years, or 18 to 60 years of age. In certain embodimentsthe subject is 1 to 17 years of age, or less than 9 years of age, orless than 4 years of age or less than 2 years of age. In furtherembodiments, the subjects or subject population are adults of more than21 years, or 21 to 60 years, or 21 to 45 years of age. According to someof these embodiments the subject is seronegative and from adengue-endemic region.

In certain embodiments, the invention is directed to said use, whereinthe unit dose of the invention as described herein is administered to apediatric subject of less than 2 years of age, preferably of 2 months to2 years of age or 2 months to 1.5 years of age or 2 months to 1 year ofage. According to some of these embodiments, the pediatric subject isseronegative and from a dengue endemic region.

In certain embodiments, the invention is directed to said use, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously to a pediatric subject of less than 2 yearsof age, preferably of 2 months to 2 years of age or 2 months to 1.5years of age or 2 months to 1 year of age. According to some of theseembodiments, the pediatric subject is seronegative and from a dengueendemic region.

In certain embodiments, the invention is directed said use, wherein thereconstituted unit dose of the invention as described herein isadministered concomitantly with another vaccine. In one embodiments, thereconstituted unit dose of the invention as described herein isadministered concomitantly with a yellow fever vaccine, in particularYF-17D. It is particularly preferred that the reconstituted unit dose ofthe invention as described herein is administered concomitantly with ayellow fever vaccine, in particular YF-17D, as described in the previoussection.

In certain embodiments, the invention is directed said use, wherein thereconstituted unit dose of the invention as described herein isadministered concomitantly with another vaccine. In one embodiments, thereconstituted unit dose of the invention as described herein isadministered concomitantly with a HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9. It is particularly preferred that thereconstituted unit dose of the invention as described herein isadministered concomitantly with a HPV vaccine, in particular a 9vHPVvaccine, such as GARDASIL® 9, as described in the previous section.

In certain embodiments, the invention is directed said use, wherein thereconstituted unit dose of the invention as described herein isadministered concomitantly with another vaccine. In one embodiments, thereconstituted unit dose of the invention as described herein isadministered concomitantly with a MMR vaccine, such as M-M-R® II. It isparticularly preferred that the reconstituted unit dose of the inventionas described herein is administered concomitantly with a MMR vaccine,such as M-M-R® II, as described in the previous section.

In certain embodiments, the invention is directed said use, wherein thereconstituted unit dose of the invention as described herein isadministered concomitantly with another vaccine. In one embodiments, thereconstituted unit dose of the invention as described herein isadministered concomitantly with a DTaP/IPV/Hib vaccine, in particular acombined DTaP/IPV/Hib vaccine, such as Pentacel®. It is particularlypreferred that the reconstituted unit dose of the invention as describedherein is administered concomitantly with a DTaP/IPV/Hib vaccine, inparticular a combined DTaP/IPV/Hib vaccine, such as Pentacel®, asdescribed in the previous section.

The present invention is in part directed to the use of thereconstituted unit dose of the invention as described herein for themanufacture of a medicament for stimulating an immune response to allfour serotypes of dengue virus in an elderly subject. In one embodimenta reconstituted unit dose of the invention as described herein isadministered by subcutaneous injection.

In certain embodiments, the invention is directed to said use, whereinthe immune response to all four serotypes of dengue virus is balanced.

In certain embodiments, the invention is directed to said use, whereinthe reconstituted unit dose is administered by subcutaneous injection tothe arm, preferably to the deltoid region of the arm.

In certain embodiments, the invention is directed the reconstituted unitdose of the invention as described herein for said use, wherein theelderly subject is seronegative to all dengue serotypes.

In certain embodiments, the invention is directed to said use, whereintwo reconstituted unit doses of the invention as described herein areadministered. In some embodiments, the two reconstituted unit doses areadministered within 12 months or more, or within six months, or withinthree months, such as at day 0 and day 90. According to some of theseembodiments, a third reconstituted unit dose is administered 6 to 12months after the administration of the first reconstituted unit dose,such as 12 months after the first administration, or later than 12 monthafter the first administration, such as 12 months (1 year) after thesecond administration or even 5 years or longer after the first orsecond administration.

In certain embodiments, the invention is directed to said use, whereinthe elderly subject is from a dengue endemic region. In otherembodiments, the elderly subject is from a dengue non-endemic region.Such an elderly subject may be subject to a vaccination according to theinvention in the context of traveling to a dengue endemic region. Incertain embodiments, the reconstituted unit dose of the invention asdescribed herein is administered subcutaneously to an elderly subjectthat is from a dengue endemic region or a dengue non-endemic region.

In certain embodiments, the invention is directed to said use, whereinthe reconstituted unit dose of the invention as described herein isadministered subcutaneously to an elderly subject that is seronegativewith respect to all dengue serotypes. In other embodiments, the elderlysubject is seropositive with respect to at least one dengue serotype.

In certain embodiments, the invention is directed to said use, whereinthe elderly subject has at least one chronic condition or disease. Theat least one chronic condition or disease may be selected from diabetes,hypertension, allergies, previous strokes, ischemic heart disease,chronic renal impairment and chronic obstructive pulmonary disease.

In certain embodiments, the invention is directed to said use, whereinthe elderly subject has an impaired immune system.

Method for Determining the Titer of Neutralizing Antibodies

The present disclosure provides a method for determining the titer ofneutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4in a blood serum sample, the method comprising the steps of:

-   -   (a) seeding cells from a dengue-susceptible cell line on 96-well        assay plates and culturing the cells for a culture period;    -   (b) preparing serial dilutions of the blood serum sample;    -   (c) separately mixing the serially diluted blood serum samples        prepared in step (b) with dengue serotype 1, dengue serotype 2,        dengue serotype 3 and dengue serotype 4 to obtain separate        mixtures for each dengue serotype and incubating the separate        mixtures;    -   (d) adding the separate mixtures prepared in (c) to the cells        seeded and cultured in step (a) and incubating the cells with        the separate mixtures;    -   (e) providing an overlay for the inoculated cells and incubating        the cells for an incubation period of 40 to 75 hours;    -   (f) determining the number of plaques in each well and comparing        the number of plaques in each well to a control to determine the        level of neutralizing antibodies against each of dengue        serotypes 1, 2, 3 and 4.

Different incubation periods may be used in step (e) for the mixtures ofdifferent dengue serotypes. The incubation period for mixtures of dengueserotype 4 may be shorter than the incubation period for mixtures ofdengue serotypes 1, 2 and 3, for example the incubation period formixtures of dengue serotype 4 may be less than 50 hours, preferably 46±2hours. In some embodiments, the incubation period for mixtures of dengueserotype 2 may be longer than the incubation period for mixtures ofdengue serotypes 1, 3 and 4, for example the incubation period formixtures of dengue serotype 2 may be between 60 and 70 hours, preferably70±2 hours.

The dengue-susceptible cell line used in step (a) may be selected fromVero cells, LLC-MK2 cells and BHK-21 cells. The culture period of thecells may be 12 to 36 hours.

In step (c) the dengue serotype 1 may be DENV-1 strain 16007, dengueserotype 2 may be DENV-2 strain 16681, dengue serotype 3 may be DENV-3strain 16562 and dengue serotype 4 may be DENV-4 strain 1036.

The separate mixtures in step (c) may be incubated overnight at atemperature of 2 to 8° C.

The overlay in step (e) may be selected from the group consisting ofmethylcellulose, carboxymethylcellulose and agarose. The cells with theoverlay may be incubated at a temperature of 33° C. to 35° C.

The number of plaques in each well may be determined usingserotype-specific anti-dengue monoclonal antibodies.

The disclosure also provides a method for determining the titer ofneutralizing antibodies against each of dengue serotypes 1, 2, 3 and 4in a blood serum sample, the method comprising the steps of:

-   -   (a) seeding Vero cells on 96-well assay plates and culturing the        Vero cells for a period of 20 to 30 hours;    -   (b) preparing serial dilutions of the serum sample;    -   (c) separately mixing the serially diluted serum samples with        dengue serotype 1, dengue serotype 2, dengue serotype 3 and        dengue serotype 4 to prepare separate mixtures and incubating        the separate mixtures overnight at a temperature of 2 to 8° C.;    -   (d) incubating the cells seeded and cultured in step (a) with        the separate mixtures prepared in step (c) in separate wells for        90 to 120 minutes;    -   (e) providing a methylcellulose overlay for the inoculated cells        and incubating the cells for an incubation period of 40 to 75        hours at 34° C.;    -   (f) determining the number of plaques in each well using        serotype-specific anti-dengue monoclonal antibodies and        comparing the number of plaques in each well to a control to        determine the level of neutralizing antibodies against each of        dengue serotypes 1, 2, 3 and 4.

The disclosure also provides the use of said method for determining thedengue serostatus of a subject before vaccination with a dengue virusvaccine or for analyzing a subjects immune response after vaccinationwith a dengue virus vaccine.

EXAMPLES

The following Examples are included to demonstrate certain aspects andembodiments of the invention as described in the claims. It should beappreciated by those of skill in the art, however, that the followingdescription is illustrative only and should not be taken in any way as arestriction of the invention.

Example 1: Preparation of the Dengue Virus Strains

The methods used to generate the chimeric dengue strains TDV-1, -3 and-4 were standard molecular cloning and DNA engineering methods and aredescribed in Huang et al. (2003) J. Virology 77(21): 11436-11447. Thefollowing well-known methods were used to construct and introduce theprM-E genes of dengue serotypes 1, 3 and 4 into the TDV-2 backbone:Reverse-transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion,DNA fragment ligation, bacterial transformations by electroporation,plasmid DNA preparations, in vitro transcription by T7 RNA polymerase,and transfection of Vero cells by electroporation.

After growing and purifying the different dengue serotypes separately asdescribed in Huang et al. (2013) PLOS Neglected Dis, 7(5):e2243, theyare mixed in the concentrations provided in Table 5. The mixture ofdengue serotypes is present in a dengue vaccine composition and combinedwith a composition of pharmaceutically acceptable excipients resultingin a dengue vaccine composition comprising 15% w/v α,α trehalosedihydrate, 1% w/v poloxamer 407, 0.1% w/v human serum albumin and 100 mMsodium chloride. The dengue vaccine composition is lyophilized andrepresents a lyophilized unit dose of TDV. The lyophilized unit dose isreconstituted with 37 mM aqueous sodium chloride solution and thereconstituted unit dose comprises 15% w/v α,α trehalose dihydrate, 1%w/v poloxamer 407, 0.1% w/v human serum albumin and 137 mM sodiumchloride.

Example 2: Microneutralization Test

Immunogenicity was measured by a microneutralization assay to each oneof the four dengue serotypes with titers defined as the dilutionresulting in a 50% reduction in plaque values (MNT50). Briefly, on day 1Vero cells were seeded on 96-well assay plates in DMEM and 10% FBS at adensity of 2.5×10⁵ cells/ml and incubated at 37° C. for 24 hours. On day2 serial dilutions of the heat-inactivated antibody-containing test andcontrol sera samples (dilutions range 1:10 to 1:20480) were prepared andmixed with a constant concentration of dengue viruses, in particularDENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain 16562 and DENV-4strain 1036, (target 60-80 pfu/well) in a 96 well microtiter plate andincubated overnight at 2-8° C. to enable the neutralization of the virusby the antibodies present in the sera. After the incubation the mixtureof virus and antibodies was transferred onto the 96 well plates withVero cells and the plates were incubated at 37° C. for 90-120 minutes toinfect the Vero cells. A 1% methylcellulose overlay in DMEM was appliedto the plate to restrict spread of progeny virus and the plate wasincubated for 46-70 hours at 34° C. depending on the Dengue serotype:

DENV1—66±2 hoursDENV2—70±2 hoursDENV3—66±2 hoursDENV4—46±2 hours

After the incubation the cells were washed twice with PBS and fixed byadding cold methanol and incubating for 60 minutes at a temperature of≤−20° C. After fixing the plates were dried and washed three times withwashing buffer (1×PBS, pH 7.4 with 0.5% Tween), before 50 μl ofserotype-specific anti-dengue monoclonal antibodies in blocking solution(2.5% nonfat dry milk in PBST) per well were added and incubated withthe cells for 18±4 hours at 2-8° C.

The monoclonal antibodies were made as described in Gentry et al. (1982)Am. J. Trop. Med. Hyg. 31, 548-555; Henchal et al. (1985) Am. J. Trop.Med. Hyg. 34, 162-169; and Henchal et al. (1982) Am. J. Trop. Med. Hyg.31(4):830-6). Briefly, the anti-DENV-1 HBD was made against dengue 1strain Hawaii, Envelope, the anti-DENV-2 was made against dengue 2strain New Guinea C, Envelope, isotype 1, the anti-DENV-3 HBD was madeagainst dengue 3 strain H87, Envelope, isotype 2A, and the anti-DENV-4HBD was made against dengue 4 strain H241, Envelope, isotype 2A.

After incubation, the plates were washed three times with washing bufferand 50 μl of a secondary peroxidase labelled goat anti-mouse IgG (H+L)(KPL Cat #074-1806) in blocking solution was added and incubated for 90to 120 minutes at 37° C. Then the plates were washed three times withwashing buffer and 50 μl of precipitant substrate (2-amino-9-ethylcarbazole (AEC) tablet in 2.5 ml DMSO, 47.5 ml 50 mM acetate buffer and250 μl hydrogen peroxide) were added and the mixture was incubated for20 minutes at room temperature. Finally, the substrate was removed, theplates were rinsed with dH₂O and dried.

Sample titers are calculated using the linear regression method andreported as MNT50 titers for each sample. Clinical data are reported asa geometric mean titer for all the individual MNT50 titers in eachtreatment group. Briefly, the number of infectious foci in each well wascounted and the titer of neutralizing antibodies was determined bycomparing the percent reduction of infectious foci centers in wellscontaining antibody (test samples) in comparison to wells containingvirus alone. The MNT50 was calculated using the following linearregression equation:

MNT50=10{circumflex over ( )}[(50−c)/m]) where c=y intercept ofregression line and m=slope of regression line

Each test sample was tested in triplicates and the titer was calculatedfrom the average of the triplicates. A schematic drawing of the stepsperformed in this test is provided in FIG. 2.

Example 3: Clinical Trial Comparing Two Different Unit Doses

A descriptive Phase II, double-blind, randomized, and controlled trialin 351 subjects aged 21 to 45 years living in Singapore was performedcomparing two different formulations of a tetravalent dengue vaccine.Subjects were randomized (1:1), stratified by baseline dengueserostatus, into two treatment groups: groups 1 received onesubcutaneous (SC) dose of HD-TDV and group 2 received one subcutaneousdose of TDV. The dengue vaccine formulations were prepared as describedin Example 1. The concentration of the four dengue serotypes in theHD-TDV vaccine (high dose tetravalent dengue vaccine) and in the TDVvaccine (corresponding to the unit dose of the invention as describedherein) is outlined in Table 5.

TABLE 5 Unit doses used in the trial. Comparative 0.5 mL comprisingTDV-1, TDV-2, TDV-3 and TDV-4 containing 3.2 × 10⁴ unit dose PFU, 1.3 ×10⁵ PFU, 2.5 × 10⁵ PFU, and 4 × 10⁵ PFU, respectively. (HD-TDV)Administered by subcutaneous injection on day 1. Example 1 0.5 mLcomprising TDV-1, TDV-2, TDV-3 and TDV-4 containing 2.5 × 10⁴ unit dosePFU, 6.3 × 10³ PFU, 3.2 × 10⁴ PFU, and 4 × 10⁵ PFU, respectively. (TDV)Administered by subcutaneous injection on day 1.

Immunogenicity was evaluated at Days 15, 30, 90, 180, and 365post-vaccination as geometric mean titers (GMTs) and seropositivityrates. Immunogenicity of the vaccines against each of the four dengueserotypes was assessed using a microneutralization assay, with titerscorresponding to the dilution resulting in a 50% reduction in plaquereduction (MNT50) as described in Example 2. Primary immunogenicityendpoints were reported in terms of geometric mean titers (GMTs) ofneutralizing antibodies, and seropositivity rates (which were defined aspercentages of subjects with reciprocal neutralizing titers 10 for eachof the DENV serotypes) in the overall trial population. As a secondaryendpoint, GMTs and seropositivity rates were analyzed by dengue baselineseropositivity status. Seropositive at baseline was defined as beingseropositive for at least one DENV serotype, whereas seronegative atbaseline was defined as being seronegative for all four DENV serotypes.

Solicited and unsolicited adverse events (AEs) were assessed by severityand causality.

a) Seropositivity

Dengue seropositivity is based on the result of the microneutralizationtest (MNT) described in Example 2 and is defined as a reciprocalneutralizing antibody titer 10 for one or more dengue serotype atbaseline. The baseline seropositivity rate for each dengue serotype isdefined as the percentage of seropositive subjects for the given dengueserotype and was derived from the neutralizing antibodies titers of thedengue serotypes as measured in the subjects before administration ofthe first unit dose. The seropositivity rate at day 180 or day 365 isdefined as the percentage of seropositive subjects and was derived fromthe neutralizing antibodies titers of the dengue serotypes as measuredin the subjects 180 and 365 days after administration of the first unitdose, respectively.

In total, 187 subjects (53.6%) were seropositive, based on MNT50, for atleast one dengue serotype at baseline: 48.7% were seropositive forDENV-1, 49.0% for DENV-2, 45.2% for DENV-3, and 41.2% for DENV-4. Theseropositive status and rate at baseline of the two differentvaccination groups is shown in Table 6.

TABLE 6 Serostatus and seropositivity rate at baseline ComparativeExample 1 unit dose unit dose Baseline seropositivity status (count ofparticipants) Seropositive for at least one dengue serotype 92 95Seronegative for all dengue serotypes 83 80 Baseline seropositivity ratefor each serotype (percentage of participants) TDV-1 48.6 48.6 TDV-247.4 50.3 TDV-3 44.0 46.3 TDV-4 41.7 40.6

Seropositivity rates increased to Day 30 after administration of theunit doses, and remained high through to Day 365 for each of the fourserotypes (FIG. 3). For the overall trial population, the percentages ofsubjects who were seropositive for DENV-1 and DENV-3 were similar in theHD-TDV and TDV groups, whereas higher post-baseline seropositivity rateswere seen for the HD-TDV group against DENV-2, and for the TDV groupagainst DENV-4 (FIG. 3B). In general, higher seropositivity rates wereseen in subjects already seropositive at baseline than in seronegativesubjects. Seropositivity rates rose to nearly 100% against all fourdengue serotypes in the seropositive vaccine groups by Day 30, andremained at this level through to Day 365; no difference was seenbetween HD-TDV and TDV (FIG. 3A). In the seronegative group, theseropositivity rates increased more gradually to a peak at Day 30, withlimited decline until Day 365. The rates were consistently higher forHD-TDV than TDV against DENV-2, but were higher for TDV than HD-TDVagainst DENV-4, through to Day 365 (FIG. 3A).

TABLE 7 Seropositivity rate at day 180 Comparative Example 1 unit doseunit dose Overall number of 166 163 participants analyzed Seropositivityrate at day 180 (95% Confidence Interval) Day 180, TDV-1 93.4 (88.5 to96.6) 89.6 (83.8 to 93.8) Day 180, TDV-2 98.2 (94.8 to 99.6) 92.6 (87.5to 96.1) Day 180, TDV-3 88.0 (82.0 to 92.5) 82.2 (75.5 to 87.7) Day 180,TDV-4 78.9 (71.9 to 84.9) 84.0 (77.5 to 89.3)

TABLE 8 Seropositivity rate at day 365 Comparative Example 1 unit doseunit dose Overall number of 160 156 participants analyzed Seropositivityrate at day 365 (95% Confidence Interval) Day 365, TDV-1 84.4 (77.8 to89.6) 89.7 (83.9 to 94.0) Day 365, TDV-2 98.8 (95.6 to 99.8) 91.0 (85.4to 95.0) Day 365, TDV-3 79.4 (72.3 to 85.4) 77.6 (70.2 to 83.8) Day 365,TDV-4 72.5 (64.9 to 79.3) 81.4 (74.4 to 87.2)

b) Geometric Mean Neutralizing Antibody Titers (GMTs)

Neutralizing antibody titers (GMTs) for each dengue serotype weredetermined in a serum sample of a subject taken before administration ofthe first unit dose of the dengue vaccine composition and 180 or 365days after administration of the first unit dose of the dengue vaccinecomposition using the microneutralization (MNT) assay as described inExample 2.

For both HD-TDV and TDV, an increase in GMTs was observed by Day 15,reaching a maximum by Day 30 (FIG. 4). Antibody titers remained abovebaseline levels throughout the trial for both unit doses. In the overalltrial population, no substantial differences were seen in GMT titersbetween the two unit dose groups, except against DENV-2, where theresponse was higher for the HD-TDV group compared with the TDV group(8640.3 versus 1992.7 at Day 30). When assessed by baselineseropositivity status, the GMT profiles were similar as for the entirepopulation, with a rise by Day 15, peak at Day 30, and gradual declinethereafter (FIG. 4B). In the group who were seropositive at baseline,the difference between the unit dose groups in response to DENV-2 wasreduced, with GMTs of 6970.3 and 4193.3 at Day 30 for the HD-TDV and TDVgroups, respectively. Responses were higher against DENV-1, DENV-3, andDENV-4 in the baseline seropositive group, compared with the baselineseronegative group, across both unit doses. Against DENV-2, a lowerresponse was seen in baseline seronegative subjects receiving TDV,compared with HD-TDV; Day 30 GMTs were 812.9 in the TDV group, comparedwith 10965.9 in the HD-TDV group. The response in these subjects alsodiffered against DENV-4, with a higher response being observed in theTDV group (Day 30 GMTs of 57.7, compared with 20.9 in the HD-TDV group);this difference persisted to Day 365 (FIG. 4A).

TABLE 9 Geometric mean neutralizing antibody titers (GMTs) at day 180Comparative Example 1 unit dose unit dose Overall number of 166 163participants analyzed GMTs (95% Confidence Interval) [units: Titer] Day180, TDV-1 379.4 (252.3 to 570.3)  312.2 (212.2 to 459.2) Day 180, TDV-22585.5 (2088.8 to 3200.3) 1235.0 (890.7 to 1712.5) Day 180, TDV-3 236.2(162.2 to 344.0)  161.0 (110.5 to 234.6) Day 180, TDV-4 91.0 (65.7 to125.9)  92.9 (68.9 to 125.4)

TABLE 10 Ratio of geometric mean neutralizing antibody titers (GMTs) atday 180 Comparative Example 1 unit dose unit dose TDV-2: TDV-1 7 4TDV-2: TDV-3 11 8 TDV-2: TDV-4 28 13

TABLE 11 Geometric mean neutralizing antibody titers (GMTs) at day 365Comparative Example 1 unit dose unit dose Overall number of 160 156participants analyzed GMTs (95% Confidence Interval) [units: Titer] Day365, TDV-1 247.3 (160.9 to 380.2)  264.1 (181.1 to 385.1)  Day 365,TDV-2 1726.0 (1392.6 to 2139.3) 809.5 (576.6 to 1136.4) Day 365, TDV-3163.2 (110.0 to 242.3)  132.6 (89.9 to 195.5)   Day 365, TDV-4 75.3(53.8 to 105.4)  77.0 (56.9 to 104.2) 

TABLE 12 Ratio of geometric mean neutralizing antibody titers (GMTs) atday 365 Comparative Example 1 unit dose unit dose TDV-2: TDV-1 7 3TDV-2: TDV-3 11 6 TDV-2: TDV-4 23 11

TABLE 13 Geometric mean neutralizing antibody titers (GMTs) of all fourdengue serotypes assessed by dengue baseline seropositivity status atday 180 Comparative unit dose Example 1 unit dose Seropositive 89Participants 88 Participants Day 180, TDV-1 (Seropositive) 2327.2(1550.4 to 3493.3) 1356.2 (905.5 to 2031.2) Day 180, TDV-2(Seropositive) 4412.0 (3586.6 to 5427.4) 2952.0 (2358.2 to 3695.4) Day180, TDV-3 (Seropositive) 1248.3 (879.7 to 1771.3)  693.6 (459.6 to1046.6) Day 180, TDV-4 (Seropositive)  399.5 (291.3 to 547.9)  268.3(190.2 to 378.6) Seronegative 77 Participants 75 Participants Day 180,TDV-1 (Seronegative)  46.6 (32.0 to 67.9)  55.7 (35.6 to 87.1) Day 180,TDV-2 (Seronegative) 1394.1 (983.2 to 1976.6)  444.3 (247.2 to 798.5)Day 180, TDV-3 (Seronegative)  34.5 (23.4 to 50.7)  29.0 (19.4 to 43.3)Day 180, TDV-4 (Seronegative)  16.4 (12.3 to 22.0)  26.8 (19.0 to 37.7)

TABLE 14 Ratio of geometric mean neutralizing antibody titers (GMTs)assessed by dengue baseline seropositivity status at day 180 and 365Comparative Example 1 unit dose unit dose Seropositive 180 DaysTDV-2:TDV-1 1.9 2.2 TDV-2:TDV-3 3.5 4.3 TDV-2:TDV-4 11.0 11.0Seronegative 180 Days TDV-2:TDV-1 29.9 8.0 TDV-2:TDV-3 40.4 15.3TDV-2:TDV-4 85.0 16.6

TABLE 15 Geometric mean neutralizing antibody titers (GMTs) of all fourdengue serotypes assessed by dengue baseline seropositivity status atday 365 Comparative unit dose Example 1 unit dose Seropositive 84Participants 85 Participants Day 365, TDV-1 (Seropositive) 1633.3(1055.8 to 2526.7) 1081.5 (724.0 to 1615.6) Day 365, TDV-2(Seropositive) 3316.0 (2623.8 to 4190.9) 2177.3 (1613.5 to 2938.1) Day365, TDV-3 (Seropositive)  830.6 (551.2 to 1251.5)  600.2 (402.3 to895.3) Day 365, TDV-4 (Seropositive)  346.3 (249.2 to 481.1)  212.6(152.2 to 296.9) Seronegative 76 Participants 71 Participants Day 365,TDV-1 (Seronegative)  30.7 (20.4 to 46.2)  48.8 (32.1 to 74.2) Day 365,TDV-2 (Seronegative)  838.7 (621.9 to 1131.1)  247.6 (143.9 to 426.1)Day 365, TDV-3 (Seronegative)  27.0 (17.8 to 41.1)  21.7 (14.3 to 33.1)Day 365, TDV-4 (Seronegative)  13.9 (10.3 to 19.0)  22.9 (15.8 to 33.1)

TABLE 16 Ratio of geometric mean neutralizing antibody titers (GMTs)assessed by dengue baseline seropositivity status at day 365 ComparativeExample 1 unit dose unit dose Seropositive 365 Days TDV-2:TDV-1 2.0 2.0TDV-2:TDV-3 4.0 3.6 TDV-2:TDV-4 9.6 10.2 Seronegative 365 DaysTDV-2:TDV-1 27.3 5.1 TDV-2:TDV-3 31.1 11.4 TDV-2:TDV-4 60.3 10.8

c) Safety

Overall, rates of solicited local and systemic adverse events (AEs),unsolicited AEs, and serious adverse events (SAEs) were similar acrossthe two unit dose groups. No deaths or AEs leading to discontinuationwere recorded in the trial. Three subjects in each unit dose groupexperienced SAEs, one of these events in the HD-TDV group was consideredby the sponsor to be vaccine-related based on temporal association. TheSAE was polyarthritis which began six days following receipt of thevaccine. Rates of solicited reactions were similar across unit dosegroups, and seropositivity status at baseline. Overall, 47.4% and 53.7%of subjects reported local reactions, and 52.0% and 50.9% reportedsolicited systemic AEs, in the HD-TDV and TDV groups, respectively. Mostreactions were mild or moderate. The most commonly reported localadverse reaction was injection site pain (46.3% in the HD-TDV group,52.0% in the TDV group) and the most common systemic AE was headache(28.6% in the HD-TDV group, 34.9% in the TDV group).

d) Conclusion

Both unit doses showed an acceptable safety profile. The results show amore balanced immune response with the new TDV unit dose compared to theearly HD-TDV unit dose, particularly in the subjects who wereseronegative prior to vaccination: (i) in baseline seronegativesubjects, response to DENV-2 was less dominant with TDV and (ii) DENV-4seropositivity rates and GMTs were also higher with TDV in thesesubjects.

Example 4: Cell-Mediated Immunity Stimulated by the Dengue Vaccine

A gamma interferon (IFNγ) enzyme-linked immunosorbent spot (ELISPOT)assay was performed using peripheral blood mononuclear cells (PBMCs)from the subjects taking part in the clinical trial of Example 3 and thecommercial ELISpot assay kit available from Mabtech according to themanufacturer's instructions. Briefly, cryopreserved PBMCs were thawedand left to rest overnight, then incubated with various peptide poolsfor 18-22 hours in plates coated with anti-IFNγ antibodies. PBMCs werethen removed and spots were developed and then counted. Results werereported as mean spot forming cells (SFC) per 10⁶ PBMCs. Peptide poolsmatched selected DENV-derived proteins, covering the entire DENV-2proteome with NS1, NS3, and NS5 proteins from New Guinea C (NGC) andThailand/16681/84 strains; and C, prM+E, NS2 and NS4 proteins fromThailand/16681/84 only plus TDV-1, TDV-3 and TDV-4 prM+E inserts fromDENV-1, -3 and -4 strains Thailand/16007/1964, Philippines/16562/1964and Indonesia/1036/1976, respectively.

Response rates to DENV-2 proteome at 6 and 12 months post-single dose ofTDV were >90%, and were comparable between subjects seronegative andseropositive at baseline (FIG. 5).

The response was primarily directed to the NS proteins, particularly insubjects seronegative at baseline (FIG. 6).

The NS3 and NS5 proteins were the most recognized antigens (by 50-75% ofsubjects). Immunodominance of NS3 and NS5 was independent of baselineserostatus. Durability of the response was maintained equally betweenNS3 and NS5 throughout the 12-month post-single vaccination follow-up.

Example 5: Antibody Responses to Non-Structural Proteins

The non-structural protein NS-1 from all four dengue serotypes caninduce endothelial hyperpermeability of human pulmonary microvascularendothelial cells (HPMEC) as measured by transendothelial electricalresistance (TEER) (Puerta-Guardo et al. (2016) PloS Pathog.12(7):e1005738). It also interacts with endothelial cells to inducedegradation of the glycocalyx via activation of sialidases and thecathepsin L/heparanase pathway (Glasner et al. (2017) PloS Pathog.13(11): e1006673). In view of these effects, it was investigated whetherthe comparative unit dose induces antibodies against NS1 and inhibitsNS1-mediated physiological effects.

a) Anti-NS1 Antibodies

Serum samples were collected at day 0 before vaccination and day 120after administration of the first unit dose. Serum was collected from 6dengue seronegative and 6 dengue seropositive subjects at both day 0 andday 120, and Ab concentrations were determined by ELISA.

The anti-NS1 antibody concentration in seronegative and seropositivesubjects at day 0 and day 120 is shown in Tables 17 and 18:

TABLE 17 Anti-NS1 antibody concentration in seronegative subjects at day0 and day 120 Dengue seronegative subjects Anti-NS1 antibodyconcentration (RU/ml) DENV1 DENV2 DENV3 DENV4 Subj. d0 d120 d0 d120 d0d120 d0 d120 1023014 13.49 602.56 16.22 2570.40 10.00 489.78 28.18302.00 1025011 66.07 173.78 35.48 794.33 67.61 117.49 42.66 85.111025013 5.62 380.19 24.55 2454.71 16.98 316.23 10.00 186.21 103500234.67 177.83 31.62 977.24 17.78 114.82 19.05 44.67 1035005 50.12 467.7420.42 1659.59 104.71 309.03 66.07 288.40 1035001 40.74 186.21 52.48489.78 44.67 169.82 51.29 177.83

TABLE 18 Anti-NS1 antibody concentration in seropositive subjects at day0 and day 120 Dengue seropositive subjects Anti-NS1 antibodyconcentration (RU/ml) DENV1 DENV2 DENV3 DENV4 Subj. d0 d120 d0 d120 d0d120 d0 d120 1052010 691.83 11481.54 309.03 12022.64 436.52 7585.78245.47 4677.35 1052014 758.58 1445.44 407.38 891.25 758.58 1122.02724.44 707.95 1052015 3890.45 3467.37 2570.40 2344.23 3235.94 2818.38660.69 707.95 1071007 478.63 851.14 239.88 478.63 660.69 1202.26 870.961258.93 1071012 691.83 776.25 724.44 676.08 776.25 812.83 346.74 446.681082009 5888.44 5370.32 7413.10 6309.57 5248.07 4897.79 891.25 794.33

These data show that the vaccine induces antibodies against NS1 from alldengue serotypes in both seropositive and seronegative subjects.

b) Transendothelial Electrical Resistance (TEER)

The effect of recombinant NS1 proteins from dengue serotypes 1, 2, 3 and4 and sera from vaccinated seronegative and seropositive subjects onendothelial permeability was evaluated by measuring TEER of HPMEC grownon a 24-well Transwell polycarbonate membrane system (Transwell®permeable support, 0.4 pM, 6.5 mm insert; Corning Inc.) as previouslydescribed (Beatty et al. (2015) Sci. Transl. Med. 7(304): 304ra141;Puerta-Guardo et al. (2016) PloS Pathog. 12(7):e1005738). Briefly, TEERwas measured in Ohms (Ω) at sequential 2-hour time-points following theaddition of test proteins using an Epithelial Volt Ohm Meter (EVOM) with“chopstick” electrodes (World Precision Instruments). Untreatedendothelial cells grown on Transwell inserts were used as negativeuntreated controls, and inserts with medium alone were used for blankresistance measurements. Relative TEER represents a ratio of resistancevalues (Ω) as follows: (Ω experimental condition−Q medium alone)/(Ωnon-treated endothelial cells−Ω medium alone). After 24 hours oftreatment, 50% of upper and lower chamber media was replaced by freshendothelial cell medium. For experiments using sera, 30 μl of culturesupernatant was removed from the apical chamber and replaced with 30 μlof serum samples immediately before the addition of 5 μg/ml DENV-2 NS1.

Day 0 serum samples from seronegative subjects did not protect againstNS1-mediated barrier dysfunction, but day 120 samples from allseronegative subjects blocked decreases in TEER values induced by NS1(see FIG. 7A). Day 0 samples from seropositive subjects produced varyinglevels of protection, and all day 120 samples from seropositive subjectscompletely abrogated NS1-induced hyperpermeability (see FIG. 7B).

c) Degradation of Glycocalyx-Like Layer (EGL)

Microscopy was performed as previously described (Puerta-Guardo et al.(2016) PloS Pathog. 12(7):e1005738). For imaging experiments, HPMEC weregrown on coverslips coated with 0.2% gelatin (Sigma) and imaged on aZeiss LSM 710 Axio Observer inverted fluorescence microscope equippedwith a 34-channel spectral detector. Images acquired using the Zen 2010software (Zeiss) were processed and analyzed with ImageJ software. AllRGB images were converted to grayscale, then mean grayscale values andintegrated density from selected areas were taken, along with adjacentbackground readings, and plotted as mean fluorescence intensity (MFI).To assess the effect of sera from vaccinated subjects on DENV2NS1-induced EGL disruption, the distribution of sialic acid and heparansulfate was examined on confluent HPMEC monolayers treated with DENV2NS1 (5 μg/ml)+negative control serum (30 μl), NS1+positive control serum(30 μl), or NS1+serum from vaccinated subjects (30 μl) and fixed with 4%paraformaldehyde (PFA) at 6 hours post-treatment. Primary antibodies(Wheat germ agglutinin (WGA) lectin conjugated to Alexa Fluor 647(WGA-A647, Molecular Probes) to stain N-acetyl neuraminic acid (sialicacid); Ab Heparan Sulfate, purified (clone F58-10E4, Amsbio) wereincubated overnight at 4° C., and detection was performed usingsecondary species-specific anti-IgG or anti-IgM antibodies conjugated toAlexa fluorophores (488 and 647).

Day 0 sera from seronegative subjects had no substantial protectiveeffect, while day 120 sera from seronegative subjects completely blockeddegradation of both sialic acid and heparan sulfate. Similarly, day 0samples from seropositive subjects exhibited varying levels ofprotection, and sera from seropositive subjects at day 120 werecompletely protective (see FIG. 8). Positive control serum was used as abaseline for protection, and negative control serum represented maximumNS1-mediated disruption. These results show that the anti-NS1 antibodyresponse stimulated by the dengue vaccine can protect againstNS1-induced hyperpermeability by preventing the degradation of key EGLcomponents.

Taken together, these results suggest that the dengue vaccine stimulatesrobust and protective anti-DENV2 NS1 Ab responses following vaccination.

Example 6: Phase III Clinical Trial in Children

A Phase III, double-blind, randomized, and placebo-controlled trial in20100 subjects aged 4 to 16 years living in Thailand, Sri Lanka,Philippines, Panama, Nicaragua, Dominican Republic, Colombia or Brazilwas performed evaluating the efficacy, safety and immunogenicity of atetravalent dengue vaccine referred to hereinafter as TDV. The trialincludes 3 parts. Part 1 evaluates vaccine efficacy (VE) and lasts untilboth of the following 2 criteria are fulfilled: (i) 120 cases of denguefever are confirmed and (ii) minimum duration of subject follow-up of 12months post-second vaccination. Part 2 is for an additional 6 months toevaluate VE and for secondary efficacy analyses. Part 3 will evaluatelong-term safety by following participants for side effects and willlast an additional 3 years.

Part 1: Active surveillance for the primary assessment of efficacy inall subjects. During this time subjects were contacted at least weeklyto ensure identification of febrile illness that could potentially bedue to dengue. This part commenced on the day of vaccination andfinished once both of the following 2 criteria were fulfilled: (i) 120cases of dengue fever are confirmed and (ii) minimum duration of subjectfollow-up of 12 months post-second vaccination. The end of Part 1 wasdefined for each subject so that the duration of follow up after thesecond vaccination was approximately the same for all subjects.Virologically-confirmed cases in Part 1 count towards the primaryefficacy objective if occurring at least 30 days post-secondvaccination. Part 1 was finished 12 months post-second vaccination

Part 2: Active surveillance for an additional 6 months for each subjectfollowing the completion of Part 1, I, i.e. 18 month post secondvaccination. During this time subjects were contacted at least weekly toensure identification of febrile illness that could potentially be dueto dengue. Virologically-confirmed cases in Parts 1 and 2 contributetowards the secondary efficacy objectives.

Part 3: Modified active surveillance for the assessment of safety in allsubjects following the completion of Part 2 and lasting 3 years for eachsubject. The modified surveillance during Part 3 will maintain at leastweekly contacts through Part 3 of the trial, but the intensity ofinvestigation will be modified based on the need for hospitalization.Surveillance will identify febrile illness of any severity that couldpotentially be due to dengue.

Criteria for Inclusion include:

-   -   The subject was aged 4 to 16 years inclusive, at the time of        randomization.    -   Individuals who were in good health at the time of entry into        the trial as determined by medical history, physical examination        (including vital signs) and clinical judgment of the        Investigator.    -   The subject and/or the subject's parent/guardian signed and        dated an assent/written informed consent form where applicable,        and any required privacy authorization prior to the initiation        of any trial procedures, after the nature of the trial has been        explained according to local regulatory requirements.    -   Individuals who can comply with trial procedures and are        available for the duration of follow-up.

Exclusion criteria include:

-   -   1. Febrile illness (temperature 38° C.) or moderate or severe        acute illness or infection at the time of randomization.    -   2. History or any illness that, in the opinion of the        Investigator, might interfere with the results of the trial or        pose an additional risk to the subject due to participation in        the trial, including but not limited to:        -   a. Known hypersensitivity or allergy to any of the vaccine            components.        -   b. Female subjects (post-menarche) who are pregnant or            breastfeeding.        -   c. Individuals with any serious chronic or progressive            disease according to judgment of the Investigator (e.g.,            neoplasm, insulin-dependent diabetes, cardiac, renal or            hepatic disease, neurologic or seizure disorder or            Guillain-Barré syndrome).        -   d. Known or suspected impairment/alteration of immune            function, including:            -   i. Chronic use of oral steroids (equivalent to 20 mg/day                prednisone ≥12 weeks/≥2 mg/kg body weight/day prednisone                ≥2 weeks) within 60 days prior to Day 1 (Month 0) (use                of inhaled, intranasal, or topical corticosteroids is                allowed).            -   ii. Receipt of parenteral steroids (equivalent to 20                mg/day prednisone ≥12 weeks/≥2 mg/kg body weight/day                prednisone ≥2 weeks) within 60 days prior to Day 1                (Month 0).            -   iii. Administration of immunoglobulins and/or any blood                products within the 3 months prior to Day 1 (Month 0) or                planned administration during the trial.            -   iv. Receipt of immunostimulants within 60 days prior to                Day 1 (Month 0).            -   v. Immunosuppressive therapy such as anti-cancer                chemotherapy or radiation therapy within 6 months prior                to Day 1 (Month 0).            -   vi. Human Immunodeficiency Virus (HIV) infection or                HIV-related disease.            -   vii. Genetic immunodeficiency.    -   3. Receipt of any other vaccine within 14 days (for inactivated        vaccines) or 28 days (for live vaccines) prior to Day 1        (Month 0) or planning to receive any vaccine within 28 days        after Day 1 (Month 0).    -   4. Participation in any clinical trial with another        investigational product 30 days prior to Day 1 (Month 0) or        intent to participate in another clinical trial at any time        during the conduct of this trial.    -   5. Previous participation in any clinical trial of a dengue        candidate vaccine, or previous receipt of a dengue vaccine.    -   6. First degree relatives of individuals involved in trial        conduct.    -   7. Females of childbearing potential who are sexually active,        and who have not used any of the acceptable contraceptive method        for at least 2 months prior to Day 1 (Month 0).    -   8. Females of childbearing potential who are sexually active,        and who refuse to use an acceptable contraceptive method up to 6        weeks post-second vaccination.    -   9. Deprived of freedom by administrative or court order, or in        an emergency setting, or hospitalized involuntarily.    -   10. Current alcohol abuse or drug addiction that may interfere        with the subject's ability to comply with trial procedures.    -   11. Identified as an employee of the Investigator or trial        center, with direct involvement in the proposed trial or other        trials under the direction of that Investigator or trial center.

Eligible subjects were randomized (2:1) into two treatment groups:groups 1 received one subcutaneous (SC) dose of TDV in the upper arm onDay 1 and on Day 90, and group 2 received one subcutaneous dose ofplacebo in the upper arm on Day 1 and on Day 90. Randomization wasstratified by region (Asia Pacific and Latin America) and age range(children aged 4-5 years, 6-11 years, and 12-16 years) to ensure eachage range has the appropriate ratio of TDV to placebo in each region.After randomization dropouts were not replaced. Study Day 1 is definedto be the date of the first dose administration of TDV or placebo. TheTDV was prepared as described in Example 1. Each subcutaneous dose ofTDV was 0.5 mL and the concentration of the four dengue serotypes in theTDV vaccine in each dose was 3.6 log₁₀ PFU/dose, 4.0 log₁₀ PFU/dose, 4.6log₁₀ PFU/dose and 5.1 log₁₀ PFU/dose of TDV-1, TDV-2, TDV-3 and TDV-4,respectively.

The “total concentration in pfu/0.5 ml” which serves as a base value forthe calculation of the percentage concentration for each individualcomponent of a tetravalent dengue vaccine is shown for one exemplarytetravalent vaccine composition comprising dengue serotype 1 in aconcentration of 3.60 log₁₀ pfu/0.5 ml, a dengue serotype 2concentration of 4.00 log₁₀ pfu/0.5 ml, a dengue serotype 3concentration of 4.60 log 10 pfu/0.5 ml and a dengue serotype 4concentration of 5.11 log₁₀ pfu/0.5 ml.

Primarily, the logarithmic values of the concentrations are convertedinto numerical values. The results of this conversion are 4×10³ pfu/0.5ml for serotype 1, 1×10⁴ pfu/0.5 ml for serotype 2, 4×10⁴ pfu/0.5 ml forserotype 3 and 1.3×10⁵ pfu/0.5 ml for serotype 4. The totalconcentration in pfu/0.5 ml is the sum of the preceding numerical valuesresulting in 1.84×10⁵ pfu/0.5 ml.

The “percentage concentration” for each of the serotypes 1, 2, 3 and 4is obtained by dividing the numerical concentration value (expressed aspfu/0.5 ml) of an individual serotype by the total concentration(expressed in pfu/0.5 ml) and multiplying the result by 100 i.e.:

Percentage concentration of serotype 1=(4×10³ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=2%

Percentage concentration of serotype 2=(1×10⁴ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=5%

Percentage concentration of serotype 3=(4×10⁴ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=22%

Percentage concentration of serotype 4=(1.3×10⁵ pfu/0.5 ml÷1.84×10⁵pfu/0.5 ml)×100=71%.

The percentage concentrations are rounded to whole numbers.

Primary Outcome Measures included the vaccine efficacy (VE) of two dosesof TDV in preventing virologically-confirmed dengue (VCD) fever inducedby any dengue serotype [time frame: 30 days post-second vaccination (Day120) until the end of Part 1]. VE is defined as 1−(λv/λc), wherein λvand λc denote the hazard rates for the TDV and placebo groups,respectively. A virologically-confirmed dengue case is defined asfebrile illness (defined as temperature ≥38° C. on any 2 of 3consecutive days) or illness clinically suspected to be dengue by theInvestigator with a positive serotype-specific reverse transcriptasepolymerase chain reaction (RT-PCR). A febrile illness will require aninterval of at least 14 days from a previous febrile illness to avoidoverlap of acute and convalescent visits from one episode with thosefrom a second episode.

Secondary Outcome Measures Include:

-   -   1) VE of two doses of TDV in preventing virologically-confirmed        dengue fever induced by each dengue serotype [time frame: from        30 days post-second vaccination (Day 120) until the end of Part        2].    -   2) VE of two doses of TDV in preventing virologically-confirmed        dengue fever induced by any dengue serotype in participants        dengue seronegative at baseline [time frame: from 30 days        post-second vaccination (Day 120) until the end of Part 2 (up to        21 months)].    -   3) VE of two doses of TDV in preventing virologically-confirmed        dengue fever induced by any dengue serotype in participants        dengue seropositive at baseline [time frame: from 30 days        post-second vaccination (Day 120) until the end of Part 2].    -   4) VE of two doses of TDV in preventing hospitalization due to        virologically-confirmed dengue fever induced by any dengue        serotype [time frame: from 30 days post-second vaccination        (Day 120) until the end of Part 2].    -   5) VE of two doses of TDV in preventing virologically-confirmed        severe dengue fever induced by any dengue serotype [time frame:        from 30 days post-second vaccination (Day 120) until the end of        Part 2].    -   6) Percentage of participants with solicited local injection        site adverse events (AEs) in the safety subset [time frame: Days        1 through 7 after each vaccination] and severity of solicited        local injection AEs. Solicited local AEs at injection site are        defined as pain, erythema and swelling that occurred within 7        days after each vaccination.    -   7) Percentage of participants with solicited systemic adverse        events (AEs) in the safety subset [time frame: Days 1 through 14        after each vaccination] and severity of solicited systemic AEs.        Solicited systemic AEs in children (<6 years) are defined as        fever, irritability/fussiness, drowsiness and loss of appetite        that occurred within 14 days after each vaccination. Solicited        systemic AEs in children 6 years) are defined as fever,        headache, asthenia, malaise and myalgia that occurred within 14        days after each vaccination.    -   8) Percentage of participants with any unsolicited adverse        events (AEs) in the safety subset [time frame: Days 1 through 28        after each vaccination]. Unsolicited AEs are any AEs that are        not solicited local or systemic AEs, as defined above.    -   9) Percentage of participants with serious adverse events (SAEs)        during Parts 1 and 2 [time frame: from Day 1 until the end of        Parts 1 and 2]. A serious adverse event (SAE) is any untoward        medical occurrence or effect that at any dose results in death,        is life-threatening, requires inpatient hospitalization or        prolongation of existing hospitalization, results in persistent        or significant disability/incapacity, is a congenital        anomaly/birth defect or is medically important due to other        reasons than the above mentioned criteria.    -   10) Percentage of participants with fatal SAEs and SAEs related        to study drug during the first and second half of Part 3 [time        frame: for 3 years (18 month halves) beginning at the end of        Part 2 (approximately 21 months after the first vaccination)].    -   11) Percentage of participants with a seropositive response for        each of the four dengue serotypes in the immunogenicity subset        [time frame: Day 1 and months 1, 3, 4, 9, 15 and then annually        (up to 3 years)]. Seropositive response is defined as a        reciprocal neutralizing titer 10. The four DENV serotypes are        DEN-1, DEN-2, DEN-3 and DEN-4.    -   12) Percentage of participants with a seropositive response for        multiple dengue serotypes in the immunogenicity subset [time        frame: Day 1 and months 1, 3, 4, 9, 15 and then annually (up to        3 years)].    -   13) Geometric Mean Titers (GMTs) of neutralizing antibodies for        each of the four dengue serotypes in the immunogenicity subset        [time frame: Day 1 and months 1, 3, 4, 9, 15 and then annually        (up to 3 years)]. GMTs of neutralizing antibodies will be        measured via microneutralization test (MNT) as described in        Example 2.

a) Study Population

After screening, 20,099 participants were randomized, and 20,071received at least one injection. In total, 97.4% of placebo participants(n/N: 6,521/6,698) and 97.3% of vaccinees (n/N: 13,038/13,401) completedPart 1 of the study (FIG. 9). Reasons for study withdrawals includedAEs, participants lost to follow-up, pregnancy, protocol violations, andwithdrawal by participants (or parents/guardians). Baselinecharacteristics were similar across both treatment groups (Table 19).Mean age of study participants was 9.6 years, with baselineseronegativity of 27.7%, and enrollment was broadly balanced acrossregions (46.5% in Asia, 53.5% in Latin America). The highestseronegative rate was in Panama (62.2%), followed by Sri Lanka (38.5%),Thailand (34.4%), Brazil (28.8%), Nicaragua (22.3%), Colombia (15.4%),the Philippines (12.4%), and the Dominican Republic (2.8%).

TABLE 19 Baseline characteristics of study population (number, %) TDVPlacebo Total Per Protocol Set Number of Participants 12,704 6,31719,021 Mean Age (Years, SD) 9.6 (3.35) 9.6 (3.34) 9.6 (3.35) BaselineSeronegative^(a) 3,533 (27.8) 1,726 (27.3) 5,259 (27.7) Female 6,314(49.7) 3,098 (49.0) 9,412 (49.5) Male 6,390 (50.3) 3,219 (51.0) 9,609(50.5) Asia Pacific 5,896 (46.4) 2,942 (46.6) 8,838 (46.5) BaselineSeronegative^(a) 1,503 (25.5) 773 (26.3) 2,276 (25.8) Latin America6,808 (53.6) 3,375 (53.4) 10,183 (53.5) Baseline Seronegative^(a) 2,030(29.8) 953 (28.2) 2,983 (29.3) Safety Set^(b) Number of Participants13,380 6,687 20,071 Mean Age (Years, SD) 9.6 (3.36) 9.6 (3.34) 9.6(3.35) Baseline Seronegative^(a) 3,714 (27.8) 1,832 (27.4) 5,547 (27.6)Female 6,651 (49.7) 3,276 (49.0) 9,929 (49.5) Male 6,729 (50.3) 3,411(51.0) 10,142 (50.5) Safety Set of Subset^(b) Number of Participants2,663 1,329 3,993 Baseline Seronegative^(a) 740 (27.8) 369 (27.8) 1,109(27.8) ^(a)Seronegative for all serotypes; seropositive defined asreciprocal neutralizing antibody titer ≥10; SD, standard deviation.^(b)numbers of participants in TVD plus placebo groups are not equal tototal numbers shown because misallocated participants (i.e. those whoreceived both TVD and placebo due to an administrative error) are notincluded in the TDV and placebo group data.

b) Febrile Illnesses and VCD

During Part 1, 5,754 and 4,663 episodes of febrile illness were reportedin Asian and Latin American sites, respectively. Acute samples wereobtained in 99.5% and 96.6% of these cases, with 98.3% and 85.1% ofsamples taken within five days, in Asia and Latin America, respectively.There were 278 VCD cases (76 hospitalized) in the safety set during theentire Part 1 period, of which 210 (58 hospitalized) were 30 dayspost-second vaccination in the PPS (Table 20; Table 22) and wereincluded in primary endpoint analysis.

c) Distribution of VCD Included in Primary Endpoint Analysis

DENV-1 was reported in all countries with VCD and included all the 21cases in Panama. In Sri Lanka, 54 of 60 VCD were DENV-2, and 87 of 109VCD in the Philippines were DENV-3. All seven DENV-4 VCD were reportedin the Philippines. No VCD were reported in Nicaragua or the DominicanRepublic. Of the associated 58 hospitalized VCD, 43 were reported in SriLanka. A total of two severe dengue (both DENV-3) and five denguehemorrhagic fever (DHF; three DENV-2; two DENV-3) cases were reported(Table 21). These seven were also the only such cases in the entire part1 safety set.

d) Vaccine Efficacy

VE against VCD of any serotype was 80.2% (95% CI: 73.3-85.3; P<0.001). Asimilar efficacy of 81% (95% CI: 64.1-90.0) between the doses and fromfirst dose onwards in the safety set (Table 20) suggests that thevaccine was efficacious after the first dose. Exploratory analysis ofthe secondary efficacy endpoints showed a trend of differential efficacyby serotype, with the highest efficacy against DENV-2 (97.7%), followedby DENV-1 (73.7%), DENV-4 (63.2% with CI containing zero), and DENV-3(62.6%; Table 3). Overall, efficacy was similar in baselineseronegatives (74.9%) and seropositives (82.2%; FIG. 10A); however, thisvaried by serotype. Efficacy against DENV-2 was not impacted byserostatus; efficacy against DENV-1 was slightly higher in baselineseropositives (79.8%; 95% CI: 51.3-91.6) than baseline seronegatives(67.2%; 95% CI: 23.2-86.0). No efficacy was observed against DENV-3 inbaseline seronegatives (−38.7%; 95% CI: −335.7-55.8) compared tobaseline seropositives (71.3%; 95% CI: 54.2-82.0). Efficacy byserostatus could not be calculated for DENV-4 because no cases wereobserved in baseline seronegatives. In the primary endpoint timeframe ofthe PPS, only five VCD requiring hospitalization were reported in thevaccine group compared with 53 cases in the placebo group, with a VE of95.4% (95% CI: 88.4-98.2; 97.2% for baseline seronegatives and 94.4% forbaseline seropositives; Table 21; FIG. 1013), consistent with a VE of93.3% (95% CI: 86.7-96.7) in the safety set from first dose onwards.

The primary vaccine efficacy (VE) of two doses of TDV in preventingvirologically-confirmed dengue (VCD) fever induced by any dengueserotype is shown in Table 20.

TABLE 20 Vaccine efficacy of TDV in preventing virologically-confirmeddengue (VCD) fever against any serotype from 30 days post-secondvaccination until end of part 1 Per Protocol Set (PPS), i.e. 12 monthspost-second vaccination. Safety set analysis from first dose to end ofPart 1 study period, i.e. 12 months post-second vaccination Placebo TDV(PPS) n = 6317 n = 12,704 number of subject evaluated 6,316 12,700number of subjects with febrile illness 1,712 3,195 number of febrileillness cases 2,591 4,692 virologically confirmed dengue fever (n [%])149 [2.4] 61 [0.5] Person-years at risk 5,670.1 11,578.7 incidentdensity 2.6 0.5 relative risk 0.20 95% CI of relative risk (0.15, 0.27)vaccine efficacy (%) 80.2 95% CI of vaccine efficacy (73.3, 85.3)p-value for vaccine efficacy <0.001 Placebo TDV (Safety Set)* number ofsubject evaluated 6,687 13,380 virologically confirmed dengue fever (n[%]) 199 [3.0] 78 [0.6] Person-years at risk 8,072.0 16,351.5 incidentdensity 2.5 0.5 vaccine efficacy (%) 80.9 95% CI of vaccine efficacy(75.2, 85.3) Note 1: Percentage of virologically confirmed dengue (VCD)fever are based on number of subjects evaluated. Note 2: Person-years atrisks is defined as cumulative time in years until start of VCD fever oruntil end of Part 1 study period or discontinuation date, whichevercomes first. Incident density is defined as the number of cases per 100person-years at risk. Percentages are based on total number(denominator) of analysis set participants evaluated and may not beequal to the total number of participants in the per protocol analysisset. *One participant had two instances of VCD during Part 1, only thefirst VCD was included in efficacy calculation Note 3: Vaccine efficacy(VE) and 2-sided 95% CIs are estimated from a Cox proportional hazardmodel with TDV as a factor, adjusted for age and stratified by region.Note 4: Statistical significance will be concluded if the lower bound ofthe 95% CI for VE is above 25%. Since the hypotheses will be tested in aconfirmatory manner at a 2-sided significance level of 5%, thecalculated p-value should be compared with 0.025. Note 5: Relative riskis calculated as the number of events divided by the number of subjectsevaluated in the TDV group, over the number of events divided by thenumber of subjects evaluated in the placebo group.

For the efficacy evaluation shown in Table 20, a case of VCD was definedas febrile illness (defined as fever ≥38° C. on any 2 of 3 consecutivedays) with a positive serotype-specific RT-PCR (i.e., positive denguedetection RT-PCR) and occurring at any time starting from 30 dayspost-second vaccination (Day 120 [Month 4]) through the end of Part 1.The analysis was performed on the Per-Protocol Set (PPS) and Safety Set.

As used herein, the “Per-Protocol Set (PPS)” consist of all subjects inthe Full Analysis Set (FAS) consisting of all randomized subjects whoreceived at least one dose of TDV or placebo who had no major protocolviolations. Major protocol violations are not receiving both doses ofTDV or placebo administration, not receiving both doses in the correctinterval, not having the correct administration of TDV or placebo, useof prohibited medications/vaccines by the subject, the subject meets anyof the exclusion criteria of 2d, 3, 4 or 5 defined above or productpreparation error.

The p-value is obtained by solving the critical value Z in the followingequation:

Upper bound of 1-sided (1−p %) CI of HR=0.75, wherein HR is the hazardratio and defined as HR=λV/λC.

e{circumflex over ( )}[β^({circumflex over ( )}) +Z*ŜE]=0.75, whereinβ^({circumflex over ( )}) defines the treatment and ŜE the relatedstandard error.

The 1-sided p-value is 1−(area to the left of the critical value Z froma standard normal distribution). Since the hypotheses will be tested ina confirmatory manner 2-sided at a significance level of 5%, thecalculated 1-sided p-value should be compared with 0.025.

In summary in Part 1 of this study, a high vaccine efficacy of 80.2%against virologically-confirmed dengue of any serotype in children 4-16years of age was found. It included an efficacy of 74.9% in baselineseronegatives and a robust 95.4% reduction in hospitalizations. Onset ofprotection could be seen after the first dose with 81% efficacy betweendoses. Overall, these results suggest a potential benefit for eachvaccine recipient regardless of prior dengue exposure or age. Thisfinding is significant because vaccine development against dengue hasbeen challenging, especially for dengue naïve individuals, and dengueremains one of the WHO's top ten threats to global health in 2019.19Furthermore, the onset of protection after the first dose has potentialutility in the context of outbreak control or travel vaccination,offering a reduction in the risk of dengue after only one dose.

Severe forms of dengue were assessed as follows: Dengue HemorrhagicFever (DHF) as defined by the 1997 WHO definition. Severe Dengue throughthe Dengue Case Adjudication Committee. The Dengue Case AdjudicationCommittee (DCAC) consisted of four members: a voting chairperson, twovoting members, and an independent non-voting statistician. The threeDCAC voting members are all physicians and clinical dengue experts. DCACmembers are not study investigators and do not have any conflict ofinterest that would bias their review of the trial data. Allnon-hospitalized cases were considered non-severe. The DCAC severedengue case criteria applied in a blinded manner tovirologically-confirmed hospitalized dengue cases are as follows: 1)bleeding abnormality, for a case to be considered severe there needs tobe a significant intervention required in response to the bleedingepisode such as blood transfusion, nasal packing, hormonal therapy, or,bleeding occurred into critical organs such as the brain; 2) plasmaleakage, for a case to be considered severe there needs to be evidenceof both plasma leakage and functional impairment (plasma leakageincludes clinical evidence, radiological evidence, or hematocritelevated >20% above normal levels or baseline; functional impairmentdefined as shock or respiratory distress); 3) liver, for a case to beconsidered severe there needs to be evidence of both hepatitis andfunctional impairment (hepatitis defined as an aspartateaminotransferase [AST] or alanine aminotransferase [ALT]>10 upper limitof normal range [ULN]; functional impairment defined as prothrombin[PT]>1.5 ULN or hypoalbuminemia); 4) renal, serum creatinine >2.5 timesULN or requiring dialysis; 5) cardiac, abnormalities intrinsic to theheart (i.e. not resulting from intravascular volume depletion) and withevidence of functional impairment (examples of intrinsic abnormality:myocarditis, pericarditis, and myopericarditis; example of functionalimpairment: new conduction abnormality resulting in irregular heartrhythm [i.e. not transient first-degree heart block]); 6) centralnervous system, any abnormality with the exception of a simple febrileconvulsion or a brief delirium; 7) shock, all shock cases consideredsevere. At least 1 functional impairment (of criterion 3, 4, 5, 6),needs to be present but the totality of data were considered by themembers in their assessment.

Further results of part 1 and part 2 are presented in Tables 21a to c.

TABLE 21a Distribution of cases contributing to primary endpoint by perprotocol set subgroup (30 days after second vaccination until end ofPart 1, i.e. 12 months after second vaccination) TDV Placebo Vaccine TDVIncidence Placebo Incidence Efficacy Dengue Cases Density Dengue CasesDensity (95% CI) VCD cases Baseline Seropositive^(a)  41/9,165 (0.4%)0.5 110/4,587 (2.4%) 2.7 82.2% (74.5%-87.6%) Baseline Seronegative^(a) 20/3,531 (0.6%) 0.6  39/1,726 (2.3%) 2.5 74.9% (57.0%-85.4%) DENV-116/12,700 (0.1%) 0.1  30/6,316 (0.5%) 0.5 73.7% (51.7%-85.7%) DENV-2 3/12,700 (<0.1%) <0.1  64/6,316 (1.0%) 1.1 97.7% (92.7%-99.3%) DENV-339/12,700 (0.3%) 0.3  51/6,316 (0.8%) 0.9 62.6% (43.3%-75.4%) DENV-4^(d) 3/12,700 (<0.1%) <0.1   4/6,316 (<0.1%) <0.1 63.2% (−64.6%-91.8%) 4-5Years Old  13/1,619 (0.8%) 0.9    23/801 (2.9%) 3.2 72.8% (46.2%-86.2%)6-11 Years Old  34/7,009 (0.5%) 0.5  85/3,491 (2.4%) 2.7 80.7%(71.3%-87.0%) 12-16 Years Old  14/4,072 (0.3%) 0.4  41/2,024 (2.0%) 2.283.3% (69.3%-90.9%) Asia  54/5,894 (0.9%) 1.0 127/2,942 (4.3%) 4.9 79.5%(71.8%-85.1%) Latin America   7/6,806 (0.1%) 0.1  22/3,374 (0.7%) 0.784.3% (63.1%-93.3%) Hospitalized VCD cases Baseline Seropositive^(a)  4/9,165 (<0.1%) <0.1  35/4,587 (0.8%) 0.8 94.4% (84.3%-98.0%) BaselineSeronegative^(a)   1/3,531 (<0.1%) <0.1  18/1,726 (1.0%) 1.2 97.2%(79.1%-99.6%) Cases of DHF^(b) All participants  1/12,700 (<0.1%) <0.1  4/6,316 (<0.1%) <0.1 87.3% (−13.5%-98.6%) Severe VCD Cases^(c) Allparticipants  1/12,700 (<0.1%) <0.1   1/6,316 (<0.1%) <0.1 50.8%(−686.9%-96.9%) VCD, virologically-confirmed dengue; DHF, denguehemorrhagic fever ^(a)Seronegative for all serotypes; baselineseropositive defined as reciprocal neutralizing antibody titer 10 to oneor more serotypes. ^(b)VCD cases meeting WHO 1997 DHF criteria;incidence density defined as the number of cases per 100 person-years atrisk; percentages are based on total number (denominator) of perprotocol set participants evaluated. ^(c)two severe VCD were notclassified as DHF. ^(d)The number of cases identified was sufficient toprovide reasonably precise estimates of vaccine efficacy against allindividual serotypes, except DENV-4.

TABLE 21b Distribution of cases contributing to secondary endpoint byper protocol set subgroup (30 days after second vaccination until end ofPart 2, i.e. 18 months after second vaccination) TDV TDV Placebo PlaceboDengue Incidence Dengue Incidence Vaccine Efficacy Cases Density CasesDensity (95% CI) VCD cases Overall 73.3% (66.5%-78.8%) BaselineSeropositive^(a) 75 0.6 150 2.4 76.1% (68.5%-81.9%) BaselineSeronegative^(a) 39 0.8 56 2.4 66.2% (49.1%-77.5%) DENV-1 38 0.2 62 0.769.8% (54.8%-79.9%) Baseline Seropositive^(a) 21 0.2 37 0.6    72.0(52.2%-83.6%) Baseline Seronegative^(a) 17 0.3 25 1    67.8(40.3%-82.6%) DENV-2 8 <0.1 80 0.9 95.1% (89.9%-97.6%) BaselineSeropositive^(a) 7 <0.1 54 0.9    93.7 (86.1%-97.1%) BaselineSeronegative^(a) 1 <0.1 26 1.1    98.1 (85.8%-99.7%) Hospitalized VCDcases Overall 13 <0.1 66 0.8 90.4% (82.6%-94.7%) BaselineSeropositive^(a) 8 <0.1 45 0.7 91.4% (81.7%-95.9%) BaselineSeronegative^(a) 5 0.1 21 0.9 88.1% (68.5%-95.5%) VCD,virologically-confirmed dengue; ^(a)Seronegative for all serotypes;baseline seropositive defined as reciprocal neutralizing antibody titer≥ 10 to one or more serotypes.

TABLE 21c Distribution of cases contributing to secondary endpoint bysafety set (first vaccination until end of Part 2, i.e. 21 months afterfirst vaccination) TDV TDV Placebo Placebo Dengue Incidence DengueIncidence Vaccine Efficacy Cases Density Cases Density (95% CI) VCDcases Overall 75.3% (69.5%-80.0%) Overall in between^(a) 81.0%(64.1%-90.0%) Baseline Seropositive^(b) 89 0.5 187 2.3 77.2%(70.6%-82.3%) Baseline Seronegative^(b) 42 0.7 70 2.3 70.6%(56.9%-79.9%) DENV-1 41 0.2 78 0.7 73.9% (61.9%-82.1%) DENV-2 14 <0.1109 1.0 93.7% (89.0%-96.4%) Hospitalized VCD cases Overall 17 <0.1 810.7 89.7% (82.6%-93.9%) VCD, virologically-confirmed dengue; ^(a)Inbetween: VCD after first vaccination and before second vaccination.^(b)Seronegative for all serotypes; baseline seropositive defined asreciprocal neutralizing antibody titer ≥ 10 to one or more serotypes.

TABLE 21d Dengvaxia ® VCD (first vaccination until 25 months after firstvaccination (i.e.13 month after third vaccination), ITT from CYD15, 9 to16 years of age)_(a) Vaccine Efficacy (95% CI) Overall VCD 64.7%(58.7%-69.8%) Baseline Seropositive^(b) 83.7% (62.2%-93.7%) BaselineSeronegative^(b) 43.2% (−61.5%-80.0%) DENV-1 54.8% (40.2%-65.9%) DENV-250.2% (31.8%-63.6%) Overall Hospitalized VCD 80.3% (64.7%-89.5%)^(a)Luis Villar et al. Efficacy of a tetravalent dengue vaccine inChildren in Latin America: N Engl J of Med 2015 Vol. 372 No2, 113-123

Clinical signs and symptoms of virologically-confirmed dengue casesduring Part 1 study period in safety set data are shown in Table 22.

TABLE 22 Clinical signs and symptoms of virologically-confirmed denguecases during Part 1 study period (safety set data) TDV Placebo Relative(N = 13,380) (N = 6,687) Risk Number of VCD Cases 78 200 — MedianDuration of Febrile Illness (days; 95% CI)^(a) 6.0 (5.7-7.4) 6.0(5.9-6.8) — Median Duration of Fever (days; 95% CI) 4.0 (3.9-4.6) 5.0(4.5-5.0) — Number of Hospitalized VCD Cases 9 67 — Median Duration ofHospitalization (days; 95% CI) 5.0 (2.8-5.4) 5.0 (4.6-5.4) — Evidence ofBleeding (%, n/N) 3.8% (3/78) 3.5% (7/200) 1.10 Plasma Leakage (%, n/N)2.6% (2/78) 6.5% (13/200) 0.39 Plasma Leakage-Pleural Effusion (%, n/N)1.3% (1/78) 1.5% (3/200) — Plasma Leakage-Ascites (%, n/N) 1.3% (1/78)3.0% (6/200) — Plasma Leakage-Radiological Signs (%, n/N) 40.0% (2/5)19.6% (10/51) — Plasma Leakage-Hematocrit Increase ≥20% (%, n/N)^(b)3.8% (2/53) 9.5% (13/137) — Platelet Count 100 × 10⁹ (%, n/N)^(c) 6.4%(5/78) 22.0% (44/200) 0.29 Platelet Count 50 × 10⁹ (%, n/N)^(c) 3.8%(3/78) 11.0% (22/200) 0.35 ALT or AST ≥1000 U/L (%, n/N)^(c) 0% (0/78)0% (0/200) — VCD, virologically-confirmed dengue; ALT, alanineaminotransferase; AST, aspartate aminotransferase ^(a)Duration offebrile illness defined as start date of earliest symptom to end date oflatest symptom plus one day (symptoms considered include fever and anygeneral symptoms). ^(b)Hematocrit increase defined as maximum hematocritbetween Day 3 and Day 7 inclusive, from onset of fever ≥20% increaseover minimum hematocrit before Day 3 or after Day 7 from onset of fever.^(c)For platelet, ALT, and AST data, assessments within 14 days of onsetof febrile illness have been considered. N refers to number of VCD caseswith available data for the specific parameter

e) Immunogenicity

The highest geometric mean titers (GMTs) were observed against DENV-2regardless of baseline serostatus (Table 24). A very high tetravalentseropositivity rate (99.5%) in baseline seronegatives one month afterthe second dose (Tables 23 and 24) was observed.

Seropositivity rate (% of seropositive subjects) for each of the fourdengue serotypes is determined at prevaccination on Day 1 (Month 0),post-first vaccination on Day 30 (Month 1), prevaccination on Day 90(Month 3), post-second vaccination on Day 120 (Month 4), Day 270 (Month9), Day 450 (Month 15), and then annually. Seropositivity rates (%participants, 95% CI) by dengue serotype per protocol set forimmunogenicity data for Day 0, Day 30, Day 90, Day 120, and Day 270 areshown in Table 23.

Seropositivity rates (% participants, 95% CI) by dengue serotype againstthree or more serotypes (trivalent) and against all four serotypes(tetravalent) per protocol set for immunogenicity data for Day 0, Day30, Day 90, Day 120, and Day 270 are shown in Table 23. The tetravalentseropositivity rates were high (>91%) in baseline seronegatives sixmonths after second dose.

TABLE 23 Seropositivity rates (% participants, 95% CI) by dengueserotype (per protocol set for immunogenicity data) BASELINESEROPOSITIVE BASELINE SERONEGATIVE TDV Placebo TDV Placebo N = 1,816 N =902 N = 702 N = 345 DENV-1   89.1 (87.6-90.5) 90.6 (88.5-92.4)   0(0-0.5)   0 (0-1.1)   99.5 (99.1-99.8) 88.6 (86.3-90.7) 94.1 (92.0-95.8) 4.9 (2.8-7.8)   99.3 (98.8-99.6) 90.2 (88.1-92.1) 91.6 (89.3-93.5)  6.1(3.8-9.2) >99.9 (99.7-100) 90.3 (88.1-92.3) 99.5 (98.6-99.9)  8.3(5.5-11.9)   99.6 (99.1-99.8) 89.8 (87.5-91.8) 95.1 (93.0-96.6)  9.0(6.0-12.8) DENV-2   96.5 (95.6-97.3) 97.2 (95.9-98.2)   0 (0-0.5)   0(0-1.1)   99.9 (99.6-100) 93.3 (91.4-94.9) 98.6 (97.4-99.4) 10.7(7.5-14.5) >99.9 (99.7-100) 94.0 (92.2-95.5) 99.0 (98.0-99.6) 12.2(8.9-16.1)   99.9 (99.6-100) 93.6 (91.7-95.2)  100 (99.4-100) 14.7(11.0-19.1)    100 (99.8-100) 94.6 (92.8-96.1)  100 (99.4-100) 18.3(14.1-23.2) DENV-3   88.1 (86.5-89.6) 88.0 (85.7-90.1)   0 (0-0.5)   0(0-1.1)   99.8 (99.4-99.9) 87.6 (85.1-89.7) 96.1 (94.3-97.4)  4.0(2.1-6.7)   99.5 (99.1-99.8) 87.3 (84.9-89.4) 94.4 (92.5-96.0)  2.0(0.8-4.1)   99.8 (99.5-100) 87.9 (85.5-90.1)  100 (99.4-100)  5.1(2.9-8.2)   99.7 (99.4-99.9) 87.1 (84.6-89.4) 96.4 (94.6-97.7)  7.7(4.9-11.3) DENV-4   88.1 (86.5-89.6) 87.4 (85.0-89.5)   0 (0-0.5)   0(0-1.1)   99.6 (99.2-99.9) 86.6 (84.1-88.8) 90.5 (88.0-92.6)  1.8(0.7-3.9)   99.3 (98.8-99.7) 86.9 (84.5-89.0) 92.0 (89.8-93.9)  2.9(1.4-5.3) >99.9 (99.7-100) 88.3 (85.9-90.4) 99.8 (99.1-100)  4.8(2.7-7.8)   99.7 (99.3-99.9) 87.6 (85.1-89.9) 97.0 (95.4-98.2)  6.3(3.9-9.7) Three or More Serotypes   87.5 (85.9-89.0) 87.3 (84.9-89.4)  0 (0-0.5)   0 (0-1.1)   99.8 (99.5-100) 87.2 (84.7-89.4) 96.5(94.8-97.8)  1.2 (0.3-3.1)   99.7 (99.3-99.9) 87.7 (85.3-89.7) 94.9(93.0-96.4)  1.7 (0.6-3.7)   99.9 (99.6-100) 88.4 (86.0-90.5) 99.8(99.1-100)  4.2 (2.2-7.0)   99.7 (99.4-99.9) 87.3 (84.7-89.5) 97.5(96.0-98.6)  5.7 (3.3-8.9) All Four Serotypes   83.5 (81.7-85.2) 83.5(80.9-85.8)   0 (0-0.5)   0 (0-1.1)   99.1 (98.5-99.5) 82.9 (80.2-85.4)85.3 (82.4-87.9)  0.9 (0.2-2.6)   98.6 (97.9-99.1) 83.6 (81.0-86.0) 84.3(81.4-86.9)  1.4 (0.5-3.3)   99.8 (99.5-100) 85.2 (82.6-87.6) 99.5(98.6-99.9)  3.5 (1.8-6.2)   99.2 (98.7-99.6) 84.6 (81.9-87.0) 91.3(88.7-93.4)  5.3 (3.1-8.5) Seropositivity rates (% participants, 95% CI)by dengue serotype (per protocol set for immunogenicity data;seropositive defined as a reciprocal neutralizing antibody titer ≥10;baseline seronegative defined as seronegative to all serotype; baselineseropositive defined as seropositive to one or more serotypes; N refersto number of participants in the analysis set; number of participantsevaluated at each tinnepoint may vary)

Geometric mean titers (GMTs) of neutralizing antibodies(microneutralization test [MNT]) for each dengue serotype are determinedat pre-vaccination on Day 1 (Month 0), post-first vaccination on Day 30(Month 1), pre-vaccination on Day 90 (Month 3), post-second vaccinationon Day 120 (Month 4), Day 270 (Month 9), Day 450 (Month 15), and thenannually. Geometric mean titers (95% CI) by dengue serotype per protocolset for immunogenicity data for Day 0, Day 30, Day 90, Day 120, and Day270 are shown in Table 24.

TABLE 24 Geometric mean titers (95% CI) by dengue serotype (per protocolset for immunogenicity data) BASELINE SEROPOSITIVE BASELINE SERONEGATIVETDV Placebo TDV Placebo N = 1,816 N-902 N = 702 N = 345 DENV-1 Day 1 410(365-461) 445 (377-524) 5.0 (5.0-5.0) 5.0 (5.0-5.0)  Day 30 2,404(2,204-2,622) 430 (361-512) 118 (106-131) 5.8 (5.3-6.3)  Day 90 1,945(1,791-2,112) 410 (349-481) 91 (82-102) 5.9 (5.4-6.3)  Day 120 2,115(1,957-2,286) 451 (381-534) 184 (169-201) 6.3 (5.7-7.0)  Day 270 1,447(1,329-1,574) 415 (350-492) 87 (79-97) 6.3 (5.7-6.9)  DENV-2 Day 1 745(674-825) 802 (697-924) 5.0 (5.0-5.0) 5.0 (5.0-5.0)  Day 30 6,697(6,301-7,117) 744 (635-870) 6,277 (5,648-6,977) 6.6 (6.0-7.3)  Day 904,826 (4,571-5,096) 729 (629-845) 1,682 (1,544-1,834) 7.0 (6.3-7.9)  Day120 4,897 (4,646-5,163) 766 (655-896) 1,730 (1,614-1,855) 7.7 (6.7-8.8) Day 270 3,692 (3,496-3,898) 776 (665-906) 929 (856-1,010) 8.7 (7.4-10.2)DENV-3 Day 1 357 (321-398) 356 (305-415) 5.0 (5.0-5.0) 5.0 (5.0-5.0) Day 30 2,255 (2,094-2,428) 349 (298-409) 194 (173-218) 5.5 (5.2-5.9) Day 90 1,563 (1,453-1,682) 321 (277-374) 94 (85-104) 5.5 (5.1-5.9)  Day120 1,761 (1,646-1,885) 353 (301-414) 228 (212-246) 6.0 (5.4-6.6)  Day270 1,089 (1,009-1,175) 307 (261-360) 72 (66-78) 6.3 (5.7-7.0)  DENV-4Day 1 218 (198-241) 234 (203-270) 5.0 (5.0-5.0) 5.0 (5.0-5.0)  Day 301,303 (1,221-1,391) 222 (191-258) 111 (98-125) 5.4 (5.0-5.7)  Day 901,002 (940-1,069) 215 (187-248) 63 (57-70) 5.5 (5.1-5.9)  Day 120 1,129(1,066-1,196) 241 (208-280) 144 (134-155) 5.8 (5.3-6.4)  Day 270 778(730-830) 229 (197-266) 64 (59-70) 6.2 (5.6-6.9) 

Vaccine viremia is assessed by three PCRs: dengue detection RT-PCR,vaccine screening PCR and TDV sequencing in subjects with febrileillness within 30 days after each vaccination.

f) Safety

Rates of serious adverse events (SAEs) were similar in the vaccine andplacebo groups (3.1% and 3.8% of participants, respectively; Table 25).One vaccinee and four placebo recipients experienced SAEs considered tobe related to receiving blinded investigational product by theinvestigator (two experienced hypersensitivity, two were diagnosed withdengue, and one with DHF). There were five deaths during Part 1, and allwere considered unrelated to the investigational product or studyprocedures. Total rates of unsolicited AEs were similar between thevaccine and placebo groups. The most commonly (≥1% ofvaccine-recipients) reported unsolicited AEs within four weeks of anydose by preferred term were pyrexia (vaccine group 1.5%; placebo 1.4%),nasopharyngitis (vaccine 2.7%; placebo 3.0%), upper respiratory tractinfection (vaccine 2.6%; placebo 2.9%), and viral infection (vaccine1.1%; placebo 0.9%). Solicited local reactions were reported morefrequently in the vaccine group.

TABLE 25a Overview of safety data. Subjects with at least one adverseevent after any vaccine dose. Data presented as number of events(percentage of subjects; number [n] of subjects/total [N] subjects)unless otherwise stated (safety set data) TDV Placebo Safety Set N =13,380 N = 6,687 SAEs 3.1% (409/13,380) 3.8% (255/6,687)Non-IP-Related^(a) SAEs 3.0% (408/13,380) 3.8% (251/6,687)IP-Related^(a) SAEs <0.1% (1/13,380) <0.1% (4/6,687) SAEs Leading to IPWithdrawal and/or Trial Discontinuation 0.1% (18/13,380) 0.1% (8/6,687)Deaths <0.1% (4/13,380) <0.1% (1/6,687) IP-Related Deaths 0% (0/13,380)0% (0/6,687) Safety Subset N = 2,663 N = 1,329 Unsolicited AEs OccurringWithin 4 Weeks of Any Dose 18.4% (490/2,663) 18.8% (250/1,329)IP-Relateda Unsolicited AEs Occurring Within 4 Weeks of Any 1.0%(27/2,663) 1.6% (21/1,329) Dose Solicited Systemic AEs Occurring Within2 Weeks of Any Dose^(b) 42.0% (1,107/2,635) 38.0% (501/1,317)IP-Related^(a) Solicited Systemic AEs Occurring Within 2 Weeks 31.2%(821/2,635) 28.2% (371/1,317) of Any Dose Solicited Local ReactionsOccurring Within 1 Week of Any 36.7% (967/2,633) 25.7% (338/1,317)Dose^(c) AE, adverse event; SAE, serious adverse event; IP,investigational product/TDV ^(a)IP-related, defined as related to theinvestigational product as assessed by investigator ^(b)onlyparticipants with diary data available were evaluated ^(c)all injectionsite (solicited local) reactions considered to be IP-related

TABLE 25b Number of participants (%) with serious adverse events afterany vaccination during Part 1 by MedDRA (Medical Dictionary forRegulatory Activities) System Organ Class in the order of decreasingfrequency (safety set data presented by TDV and placebo group for eventsthat occurred in >3 participants due to risk of unblinding). TDV PlaceboTotal* MedDRA System Organ Class N = 13,380 N = 6,687 N = 20,071 AnySerious Adverse Events 409 (3.1) 255 (3.8) 664 (3.3) Infections andinfestations 235 (1.8) 179 (2.7) 414 (2.1) Injury, poisoning andprocedural complications  87 (0.7)  37 (0.6) 124 (0.6) Gastrointestinaldisorders  23 (0.2)  9 (0.1)  32 (0.2) Nervous system disorders  14(0.1)  6 (<0.1)  20 (<0.1) Respiratory, thoracic and mediastinaldisorders  14 (0.1)  6 (<0.1)  20 (<0.1) Renal and urinary disorders  15(0.1)  3 (<0.1)  18 (<0.1) Blood and lymphatic system disorders  8(<0.1)  2 (<0.1)  10 (<0.1) Pregnancy, puerperium and perinatalconditions  8 (<0.1)  2 (<0.1)  10 (<0.1) Skin and subcutaneous tissuedisorders  7 (<0.1)  3 (<0.1)  10 (<0.1) Psychiatric disorders  7 (<0.1) 2 (<0.1)  9 (<0.1) General disorders and administration site conditions 5 (<0.1)  3 (<0.1)  8 (<0.1) Immune system disorders  3 (<0.1)  4(<0.1)  7 (<0.1) Metabolism and nutrition disorders  6 (<0.1)  1 (<0.1) 7 (<0.1) Musculoskeletal and connective tissue  1 (<0.1)  5 (<0.1)  6(<0.1) Social circumstances  2 (<0.1)  4 (<0.1)  6 (<0.1) Congenital,familial and genetic disorders  3 (<0.1)  2 (<0.1)  5 (<0.1) Neoplasmsbenign, malignant and unspecified  3 (<0.1)  1 (<0.1)  4 (<0.1)(including cysts and polyps) Endocrine disorders — —  3 (<0.1)Hepatobiliary disorders — —  3 (<0.1) Reproductive system and breastdisorders — —  3 (<0.1) Vascular disorders — —  3 (<0.1) Cardiacdisorders — —  2 (<0.1) Eye disorders — —  2 (<0.1) Investigations — — 1 (<0.1) Product issues — —  1 (<0.1) Surgical and medical procedures ——  1 (<0.1) *Total column includes participants who received bothTAK-003 and placebo due to administration error and are excluded fromthe TAK-003 and placebo groups. N in column header refers to number ofparticipants in the safety set

TABLE 25c Number of participants (%) with unsolicited adverse events ofany severity up to 28-days after any vaccination by MedDRA System OrganClass in the order of decreasing frequency (Subset of safety set datapresented by TDV and placebo group for events that occurred in >6participants due to risk of unblinding). TDV Placebo Total MedDRA SystemOrgan Class N = 2,663 N = 1,329 N = 3,993 Any Unsolicited Adverse Events487 (18.3) 249 (18.7) 736 (18.4) Infections and infestations 368 (13.8)190 (14.3) 558 (14.0) Injury, poisoning and procedural complications  21(0.8)  22 (1.7)  43 (1.1) Gastrointestinal disorders  33 (1.2)  9 (0.7) 42 (1.1) General disorders and administration site conditions  30 (1.1) 11 (0.8)  41 (1.0) Skin and subcutaneous tissue disorders  27 (1.0)  7(0.5)  34 (0.9) Nervous system disorders  18 (0.7)  13 (1.0)  31 (0.8)Respiratory, thoracic and mediastinal disorders  18 (0.7)  10 (0.8)  28(0.7) Blood and lymphatic system disorders  6 (0.2)  5 (0.4)  11 (0.3)Musculoskeletal and connective tissue disorders  6 (0.2)  5 (0.4)  11(0.3) Immune system disorders — —  6 (0.2) Psychiatric disorders — —  3(<0.1) Reproductive system and breast disorders — —  3 (<0.1) Ear andlabyrinth disorders — —  2 (<0.1) Cardiac disorders — —  1 (<0.1)Congenital, familial and genetic disorders — —  1 (<0.1) Eye disorders ——  1 (<0.1) Renal and urinary disorders — —  1 (<0.1) Socialcircumstances — —  1 (<0.1) *Total column includes participants whoreceived both TAK-003 and placebo due to administration error andareexcluded from the TAK-003 and placebo groups. N in column headerrefers to number of participants in the subset of safety set.

TABLE 25d Summary of diary reported injection site reactions up to 7days and systemic adverse events up to 14 days after any vaccination(Subset of safety set data). Data presented as number of participantswith events/number of evaluated participants in the analysis set (% ofevaluated participants with events). Solicited Events TDV PlaceboInjection site reactions (Age <6 years) Any 106/331 (32.0)  43/169(25.4) Pain 104/331 (31.4)  43/169 (25.4) Erythema  5/331 (1.5)  1/169(0.6) Swelling  11/331 (3.3)  2/169 (1.2) Injection site reactions (Age≥6 years) Any 861/2302 (37.4) 295/1148 (25.7) Pain 853/2302 (37.1)293/1148 (25.5) Erythema  33/2301 (1.4)  1/1147 (<0.1) Swelling  33/2300(1.4)  6/1147 (0.5) Systemic adverse events (Age <6 years) Any  88/331(26.6)  35/169 (20.7) Irritability/Fussiness  41/331 (12.4)  16/169(9.5) Drowsiness  45/331 (13.6)  21/169 (12.4) Loss of Appetite  57/331(17.2)  22/169 (13.0) Fever (Body temperature >= 38° C. or 100.4° F.) 45/327 (13.8)  23/169 (13.6) Systemic adverse events (Age ≥6 years) Any941/2302 (40.9) 422/1147 (36.8) Headache 715/2302 (31.1) 326/1147 (28.4)Asthenia 404/2302 (17.5) 187/1147 (16.3) Malaise 510/2301 (22.2)226/1147 (19.7) Myalgia 554/2302 (24.1) 216/1147 (18.8) Fever (Bodytemperature >= 38° C. or 100.4° F.) 221/2279 (9.7) 124/1134 (10.9)

Example 7: Concomitant Administration of a Yellow Fever Vaccine and aDengue Vaccine

A phase 3, observer-blind, randomized, multi-center trial will beconducted in about 900 healthy adults aged 18 to 60 years in non-endemicareas for dengue disease and yellow fever to investigate theimmunogenicity and safety of the concomitant and sequentialadministration of the unit dose as described herein (TDV) and YF-17Dvaccine. Subjects will be randomized equally (1:1:1 ratio) to one of thefollowing 3 trial groups (300 subjects per trial group):

-   -   Group 1: YF-17D vaccine and placebo concomitantly administered        on day 0 (month 0), first dose of TDV administered on day 90        (month 3), and second dose of TDV administered on day 180 (month        6).    -   Group 2: first dose of TDV and placebo concomitantly        administered on day 0 (month 0), second dose of TDV administered        on day 90 (month 3), and YF-17D vaccine administered on day 180        (month 6).    -   Group 3: first dose of TDV and YF-17D vaccine concomitantly        administered on day 0 (month 0), second dose of TDV administered        on day 90 (month 3), and placebo administered on day 180 (month        6).

Concomitantly administered vaccines will be injected to opposite arms.All subjects will be followed-up for 6 months after the thirdvaccination (administered approximately 6 months after the firstvaccination), so the trial duration will be approximately 360 days (12months) for each subject.

For evaluation of the immune response to TDV and YF-17D blood sampleswill be collected and analyzed. Blood samples for the measurement ofdengue neutralizing antibodies (microneutralization test 50% (MNT50))will be collected at pre-first vaccination (Day 0 (month 0)), 1 monthpost first vaccination (Day 30 (month 1)), pre-second vaccination (Day90 (month 3)), 1 month post second vaccination (Day 120 (month 4)),pre-third vaccination (Day 180 (month 6)), and 1 month post thirdvaccination (Day 210 (month 7)). Blood samples for the measurement of YFneutralizing antibodies (plaque reduction neutralization test (PRNT))will be collected at pre-first vaccination (Day 0 (month 0)), 1 monthpost first vaccination (Day 30 (month 1)), pre-third vaccination (Day180 (month 6)), and 1 month post third vaccination (Day 210 (month 7)).

Example 8: Concomitant Administration of a Hepatitis a Vaccine and aDengue Vaccine

A randomized, observer blind, phase 3 trial will be conducted in about900 healthy adult subjects aged 18 to 60 years (distributed across theentire age range) in non-endemic countries for dengue and hepatitis Avirus (HAV) to investigate the immunogenicity and safety of 2 doses oftetravalent dengue vaccine TDV (subcutaneous (SC) injection), and of theco-administration of a single dose of HAV vaccine (intramuscular (IM)injection) and TDV (SC injection). Subjects will be randomized equally(1:1:1 ratio) to one of the following 3 trial groups (300 subjects pergroup):

-   -   Group 1: HAV vaccine (IM) and placebo (SC), co-administered at        day 0 (month 0); placebo (SC) administered at day 90 (month 3).    -   Group 2: TDV (SC) and placebo (IM), co-administered at day 0        (month 0); TDV (SC) administered at day 90 (month 3).    -   Group 3: TDV (SC) and HAV vaccine (IM), co-administered at day 0        (month 0); TDV (SC) administered at day 90 (month 3).

Co-administered trial vaccines will be injected to opposite arms. Normalsaline solution for injection (0.9% NaCl) will be used as placebo. Ablood sample for an anti-HAV antibody test will be collected atscreening from all subjects to exclude subjects who are positive foranti-HAV antibodies. All subjects will be followed-up for 6 months afterthe second vaccination at day 90 (month 3), so the trial duration willbe 270 days or 9 months for each subject (not including the screeningperiod). Outside the context of this trial, subjects in Groups 1 and 3will be offered a HAV vaccine booster dose after the completion of trialprocedures at day 270 (month 9).

Dengue neutralizing antibodies (microneutralization test (MNT50)) willbe measured using blood samples collected at pre-first trial vaccination(day 0 (month 0)), 1 month post first trial vaccination (day 30 (month1)), and 1 month post second trial vaccination (day 120 (month 4)).Blood samples for the measurement of anti-HAV antibodies (enzyme-linkedimmunosorbent assay (ELISA)) will be collected at pre-first trialvaccination (day 0 (month 0)) and 1 month post first trial vaccination(day 30 (month 1)).

The primary endpoint includes the proportion of HAV/DENV-naive subjectsat baseline who are seroprotected against HAV at day 30 (month 1) asmeasured by enzyme-linked immunosorbent assay (ELISA) (seroprotectionrate) in a subset of 120 subjects in each group (immunogenicity subset)will be determined. Seroprotection is defined as serum anti-HAV antibodylevels ≥10 mIU/mL. Immunological naivety to HAV/DENV is defined asanti-HAV antibody levels <10 mIU/mL and reciprocal neutralizing titersfor all 4 dengue serotypes <10.

The secondary endpoints include the geometric mean titers ofneutralizing antibodies (GMTs) (microneutralization test (MNT50)) foreach of the 4 dengue serotypes at day 30 (month 1) and day 120 (month 4)will be determined in HAV/DENV-naive subjects at baseline, theproportion of HAV/DENV-naive subjects at baseline who are seropositivefor each of the 4 dengue serotypes at day 30 (month 1) and day 120(month 4) (seropositivity rate), and Geometric mean concentrations (GMC)of anti-HAV antibodies at day 30 (month 1) in subjects HAV/DENV-naive atbaseline.

Seropositivity for dengue virus is defined as a reciprocal neutralizingtiter 10 for any of the four dengue serotypes.

Example 9: Concomitant Administration of a HPV Vaccine and a DengueVaccine

A phase 3, open-label, randomized, multicenter trial will be conductedin about 430 healthy females aged ≥9 to <15 years in dengue endemicregions to investigate the immunogenicity and safety of theco-administration of TDV and 9vHPV vaccine vs 9vHPV vaccine alone.Subjects will be randomized equally to 1 of 2 groups (about 215 subjectsper trial group):

-   -   Group 1: first doses of 9vHPV vaccine+TDV co-administered on day        0 (month 0), second dose of TDV administered on Day 90 (month        3), second dose of 9vHPV vaccine administered on Day 180 (month        6).    -   Group 2: first dose of 9vHPV vaccine administered on day 0        (month 0), second dose of 9vHPV vaccine administered on Day 180        (month 6).

Concomitantly administered vaccines will be injected to opposite arms.All subjects will be followed-up for 6 months after the last trialvaccination, so the trial duration will be approximately 360 days (or 12months) for each subject.

Blood samples for the measurement of HPV neutralizing antibodies (Merckassay) for both Groups 1 and 2 will be collected at pre-firstvaccination (day 0 (Month 0)) and at 1 month post-second 9vHPVvaccination (day 210 (month 7)). Blood samples for the measurement ofdengue neutralizing antibodies (by microneutralization test 50% (MNT50))will be collected for Group 1 only at pre-first vaccination (day 0(month 0)) and at 1 month post-second TDV vaccination (day 120 (month4)).

The primary endpoints includes the geometric mean titers (GMTs) for HPVTypes 6, 11, 16, 18, 31, 33, 45, 52, 58 on day 210 (month 7).

The secondary endpoints includes seropositivity rates (% of subjectsseropositive) for HPV Types 6, 11, 16, 18, 31, 33, 45, 52 and 58 on day210 (month 7) as measured by competitive Luminex immunoassay (cLIA) orequivalent assay, GMTs of neutralizing antibodies (by MNT50) for each ofthe 4 dengue serotypes on day 120 (month 4), and seropositivity rates (%of subjects seropositive) for each of the 4 dengue serotypes and formultiple (2, 3 or 4) dengue serotypes on day 120 (month 4).

Seropositivity for dengue virus is defined as a reciprocal neutralizingantibody titer 10 for any of the 4 dengue serotypes.

Seropositivity for HPV is defined as an anti-HPV titer greater than orequal to the pre-specified serostatus cut-off for a given HPV type.Seronegativity is defined as an anti-HPV titer less than thepre-specified serostatus cut-off for a given HPV type. The serostatuscut-off is the antibody titer level above the assay's lower limit ofquantification that reliably distinguishes sera samples classified byclinical likelihood of HPV infection and positive or negative status byprevious versions of cLIA or equivalent assay. The lower limits ofquantification and serostatus cut-offs for each of the 9 vaccine HPVtypes are shown below.

TABLE 26 Competitive Luminex Immunoassay Limits of Quantification andSerostatus Cutoffs for 9vHPV types cLIA Lower Limit cLIA Serostatus ofQuantification Cutoff HPV type (mMU^((a))/mL) (mMU^((a))/mL) HPV 6 16 30HPV 11 6 16 HPV 16 12 20 HPV 18 8 24 HPV 31 4 10 HPV 33 4 8 HPV 45 3 8HPV 52 3 8 HPV 58 4 8 ^((a))mMU = milli-Merck Units

Serum antibodies to HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 willbe measured with a competitive Luminex immunoassay or equivalent assay.Titers will be reported in milli Merck Units/mL with the use of theLuminex immunoassay.

Example 10: Concomitant Administration of a Tdap Vaccine and a DengueVaccine

A phase 3, open-label, randomized trial will be conducted in about 840healthy subjects aged of ≥10 to <18 years in dengue endemic areas, wherethe Tdap vaccine is licensed for children and adolescents from 10 up to18 years of age to investigate the immunogenicity and safety of theco-administration of TDV and the Tdap vaccine BOOSTRIX® vs BOOSTRIX®alone.

TDV will be administered subcutaneously and BOOSTRIX® will beadministered intramuscularly. Subjects will be randomized equally (1:1ratio) to each one of the following 2 trial groups (about 420 subjectsper trial group):

-   -   Group 1: first dose of TDV+Tdap vaccine co-administered on day 0        (month 0); and second dose of TDV administered on day 90 (month        3).    -   Group 2: Tdap vaccine administered on day 0 (month 0).

Concomitantly administered vaccines will be injected to opposite arms.All subjects will be followed up for 9 months (270 days) after the firstvaccination, so the trial duration will be approximately 270 days foreach subject.

For each subject there will be 5 scheduled clinic visits: day 0 (month0), day 30 (month 1), day 90 (month 3), day 120 (month 4), and day 270(month 9). Blood samples for the measurement of antibody response to theTdap vaccine will be collected on day 0 (month 0) and on day 30(month 1) from all subjects in Group 1 and 2. Antibodies against thefollowing antigens will be measured:

-   -   pertussis antigens (inactivated pertussis toxin (iPT),        formaldehyde-treated filamentous hemagglutinin (FHA)), and        pertactin (PRN)),    -   tetanus toxoid antigen, and    -   diphtheria toxoid antigen.

Blood samples for the measurement of dengue neutralizing antibodies (bymicroneutralization test 50% (MNT50)) will be collected from Group 1 onday 0 (month 0), day 30 (month 1), and day 120 (month 4). Blood sampleswill also be collected prior to vaccination.

The primary endpoints include:

-   (i) the proportion of subjects seroprotected for diphtheria as    measured by Neutralizing Toxin Assay (NTA) or equivalent assay on    day 30 (month 1), wherein seroprotection is defined as    anti-diphtheria antibody levels (NTA or equivalent assay) IU/mL in    serum-   (ii) The proportion of subjects seroprotected for tetanus as    measured by Enzyme Linked Immunosorbent Assay (ELISA) or equivalent    assay on day 30 (month 1), wherein seroprotection is defined as    anti-tetanus antibody levels (ELISA or equivalent assay) IU/mL in    the serum.-   (iii) Geometric mean concentration (GMC) of acellular pertussis    antibodies (anti-iPT antibodies, anti-FHA antibodies, anti-PRN    antibodies,) as measured by ELISA or equivalent assay on day 30    (month 1).

The secondary endpoints include evaluations include geometric meanneutralizing antibody titers (GMTs) of antibodies (by MNT50) for each ofthe four dengue serotypes on day 30 (month 1) following a first dosewith TDV and on day 120 (month 4) following a second dose of TDV,seropositivity rates (% of subjects seropositive) for each of the fourdengue serotypes and for multiple (2, 3 or 4) dengue serotypes on day 30(month 1) following a first dose with TDV and on day 120 (month 4)following a second dose of TDV, wherein seropositive for each dengueserotype is defined as the percentage of subjects with a reciprocalneutralizing antibody titer of ≥10.

First List of Items of the Present Invention

1. A unit dose of a dengue vaccine composition comprising:a tetravalent dengue virus composition including four live, attenuateddengue virus strains wherein the unit dose is lyophilized and uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises:

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        2. The unit dose of item 1, wherein upon reconstitution with 0.5        mL of a pharmaceutically acceptable diluent    -   (v) has a concentration of 3.3 log 10 pfu/0.5 mL to 5.0 log 10        pfu/0.5 mL,    -   (vi) has a concentration of 2.7 log 10 pfu/0.5 mL to 4.9 log 10        pfu/0.5 mL,    -   (vii) has a concentration of 4.0 log 10 pfu/0.5 mL to 5.7 log 10        pfu/0.5 mL, and    -   (viii) has a concentration of 4.5 log 10 pfu/0.5 mL to 6.2 log        10 pfu/0.5 mL.        3. The unit dose of item 1 or 2, wherein upon reconstitution        with 0.5 mL of a pharmaceutically acceptable diluent    -   (i) has a concentration of 3.3 log 10 pfu/0.5 mL to 3.6 log 10        pfu/0.5 mL,    -   (ii) has a concentration of 2.7 log 10 pfu/0.5 mL to 4.0 log 10        pfu/0.5 mL,    -   (iii) has a concentration of 4.0 log 10 pfu/0.5 mL to 4.6 log 10        pfu/0.5 mL, and    -   (iv) has a concentration of 4.5 log 10 pfu/0.5 mL or 4.6 log 10        pfu/0.5 mL to 5.1 log 10 pfu/0.5 mL.        4. The unit dose of any one of items 1 to 3, wherein upon        reconstitution with a pharmaceutically acceptable diluent (i),        (ii), (iii), and (iv) provide a total concentration of pfu/0.5        mL and based on said total concentration the concentration        of (ii) in pfu/0.5 mL is less than 10% or less than 8%, and the        concentration of (iv) in pfu/0.5 mL is at least 50%.        5. The unit dose of item 4, wherein upon reconstitution with a        pharmaceutically acceptable diluent (i), (ii), (iii), and (iv)        provide a total concentration of pfu/0.5 mL and based on said        total concentration the concentration of (i) in pfu/0.5 mL is at        least 1%, and the concentration of (iii) in pfu/0.5 mL is at        least 7% or at least 8%.        6. The unit dose of any one of items 1 to 5, wherein        reconstitution with a pharmaceutically acceptable diluent is        made with 0.5 ml of the pharmaceutically acceptable diluent.        7. The unit dose of any one of items 1 to 6, wherein the        lyophilized unit dose is prepared from a solution further        comprising a non-reducing sugar, a surfactant, a protein and an        inorganic salt.        8. The unit dose of item 7, wherein the non-reducing sugar is        trehalose, the surfactant is poloxamer 407, the protein is human        serum albumin and the inorganic salt is sodium chloride.        9. The unit dose of item 7 or 8, wherein the solution comprises:    -   from about 10% (w/v) to about 20% (w/v) α,α-trehalose dihydrate        or an equimolar amount of other forms of α,α-trehalose,    -   from about 0.5% (w/v) to about 1.5% (w/v) poloxamer 407,    -   from about 0.05% (w/v) to about 2% (w/v) human serum albumin,        and    -   from about 70 mM to about 140 mM sodium chloride.        10. The unit dose of any one of items 1 to 9, wherein the        lyophilized unit dose is prepared from a solution comprising:    -   about 15% (w/v) α,α-trehalose dihydrate,    -   about 1% (w/v) poloxamer 407,    -   about 0.1% (w/v) human serum albumin, and    -   and about 100 mM sodium chloride.        11. The unit dose of any one of items 1 to 10, wherein each one        of the four live attenuated dengue virus strains has attenuating        mutations in the 5′-noncoding region (NCR) at nucleotide 57 from        cytosine to thymine, in the NS1 gene at nucleotide 2579 from        guanine to adenine resulting in an amino acid change at position        828 from glycine to asparagine, and in the NS3 gene at        nucleotide 5270 from adenine to thymine resulting in an amino        acid change at position 1725 from glutamine to valine,        preferably further comprising one or more of the mutations        selected from the list comprising:    -   a) a mutation in the NS2A gene at nucleotide 4018 from cytosine        to thymine resulting in an amino acid at position 1308 from        leucine to phenylalanine,    -   b) a silent mutation in the NS3 gene at nucleotide 5547 from        thymine to cytosine, and    -   c) a mutation in the NS4A gene at nucleotide 6599 from guanine        to cytosine resulting in an amino acid change at position 2168        from glycine to alanine.        12. The unit dose of item 11, wherein (i) further comprises one        or more of the mutations selected from the list comprising:    -   a mutation in the NS2A gene at nucleotide 3823 from adenine to        cytosine resulting in an amino acid change at position 1243 from        isoleucine to leucine,    -   a mutation in the NS2B gene at nucleotide 4407 from adenine to        thymine resulting in an amino acid change at position 1437 from        glutamine to asparagine, and    -   a silent mutation in the NS4B gene at nucleotide 7311 from        adenine to guanine.        13. The unit dose of item 11 or 12, wherein (ii) further        comprises one or more of the mutations selected from the list        comprising:    -   a mutation in the prM gene at nucleotide 592 from adenine to        guanine resulting in an amino acid change at position 166 from        lysine to glutamine, and    -   a mutation in the NS5 gene at nucleotide 8803 from adenine to        guanine resulting in an amino acid change at position 2903 from        isoleucine to valine.        14. The unit dose of any one of items 11 to 13, wherein (iii)        further comprises one or more of the mutations selected from the        list comprising:    -   a mutation in the E gene at nucleotide 1603 from adenine to        thymine resulting in an amino acid change at position 503 from        threonine to serine, and    -   a silent mutation in the NS5 gene at nucleotide 7620 from        adenine to guanine.        15. The unit dose of any one of items 11 to 14, wherein (iv)        further comprises one or more of the mutations selected from the        list comprising    -   a silent mutation in the C gene at nucleotide 225 from adenine        to thymine,    -   a mutation in the NS2A gene at nucleotide 3674 from adenine to        guanine resulting in an amino acid change at position 1193 from        asparagine to glycine,    -   a mutation in the NS2A gene at nucleotide 3773 from adenine to        an adenine/guanine mix resulting in an amino acid change at        position 1226 from lysine to a lysine/asparagine mix,    -   a silent mutation in the NS3 gene at nucleotide 5391 from        cytosine to thymine,    -   a mutation in the NS4A gene at nucleotide 6437 from cytosine to        thymine resulting in an amino acid change at position 2114 from        alanine to valine,    -   a silent mutation in the NS4B gene at nucleotide 7026 from        thymine to a thymine/cytosine mix, and    -   a silent mutation in the NS5 gene at nucleotide 9750 from        adenine to cytosine.        16. The unit dose of any one of items 1 to 15, wherein    -   (i) has the amino acid sequence of SEQ ID NO. 2,    -   (ii) has the amino acid sequence of SEQ ID NO. 4,    -   (iii) has the amino acid sequence of SEQ ID NO. 6, and    -   (iv) has the amino acid sequence of SEQ ID NO. 8.        17. The unit dose of any one of items 1 to 16 reconstituted with        0.3 to 0.8 mL of liquid for reconstitution.        18. The unit dose of item 17 reconstituted with 0.5 mL of liquid        for reconstitution.        19. The unit dose of item 17 or 18, wherein the liquid for        reconstitution is 37 mM aqueous sodium chloride solution.        20. A kit for preparing a reconstituted unit dose comprising the        following components:        a) a unit dose of any one of items 1 to 16, and        b) a pharmaceutically acceptable diluent for reconstitution.        21. The kit of item 20, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.        22. Container, such as a vial, comprising one to ten unit doses        of any one of items 1 to 19.        23. A method of preventing dengue disease in a subject        population comprising administering to the subject population a        reconstituted unit dose of any one of items 17 to 19.        24. The method of item 23, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of said unit dose 90 days after said first        administration, provide a ratio of not more than 50, or not more        than 40, or nor more than 30, or not more than 20 for the GMT of        dengue serotype 2 to the GMT of dengue serotype 4.        25. The method of item 24, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        26. A method of preventing dengue disease in a subject        comprising administering to the subject a reconstituted unit        dose of any one of items 17 to 19.        27. The method of item 26, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of said unit dose 90 days after said first        administration, provide a ratio of not more than 50, or not more        than 40, or nor more than 30, or not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 4.        28. The method of item 27, wherein said neutralizing antibody        titers of the subject further provide a ratio of not more than        20 for the neutralizing antibody titer of dengue serotype 2 to        the neutralizing antibody titer of dengue serotype 1, and/or a        ratio of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        29. The method of any one of items 23 to 28, wherein the method        is for preventing dengue hemorrhagic fever (DHF) or dengue shock        syndrome (DSS).        30. The method of any one of items 23 to 29, wherein the        reconstituted unit dose is administered by subcutaneous        injection, preferably to the deltoid region of the arm.        31. The method of any one of items 23 to 30, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more.        32. The method of any one of items 23 to 30, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within six months, preferably within three months.        33. The method of item 32, wherein the two reconstituted unit        doses are administered at day 0 and day 90.        34. The method of any one of items 31 to 33, wherein a third        unit dose is administered after administration of the second        unit dose, preferably within 12 months after administration of        the first unit dose.        35. The method of any one of items 31 to 33, wherein a third        unit dose is administered after administration of the second        unit dose, preferably within 12 months after administration of        the second unit dose.        36. The method of any one of items 23 to 35, wherein the subject        or subject population is seronegative with respect to all dengue        serotypes.        37. The method of any one of items 23 to 35, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        38. The method of any one of items 23 to 37, wherein the subject        or subject population is of 2 to 60 years of age.        39. The method of any one of items 23 to 37, wherein the subject        or subject population is under 9 years of age, under 4 years of        age, or under 2 years of age.        40. The method of any one of items 23 to 39, wherein the subject        or subject population is from a dengue endemic region.        41. The method of any one of items 23 to 39, wherein the subject        or subject population is from a dengue non-endemic region.        42. A method for stimulating an immune response to all four        serotypes of dengue virus in a subject, comprising administering        to the subject a reconstituted unit dose of items 17 to 19.        43. The method of item 42, wherein the immune response to all        four serotypes of dengue virus is balanced.        44. The method of item 42 or 43, wherein the reconstituted unit        dose is administered by subcutaneous injection, preferably to        the deltoid region of the arm.        45. The method of any one of items 42 to 44, wherein two unit        doses of any one of items 17 to 19 are administered within 12        months or more.        46. The method of any one of items 42 to 45, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within six months, preferably within three months.        47. The method of item 46, wherein the two reconstituted unit        doses are administered at day 0 and day 90.        48. The method of any one of items 45 to 47, wherein a third        unit dose is administered after the administration of the second        unit dose, preferably within 12 month of administration of the        first unit dose.        49. The method of any one of items 45 to 47, wherein a third        unit dose is administered after the administration of the second        unit dose, preferably within 12 month of administration of the        second unit dose.        50. The method of any one of items 42 to 49, wherein the subject        is from a dengue endemic region.        51. The method of any one of items 42 to 49, wherein the subject        is from a dengue non-endemic region.        52. The method of any one of items 42 to 51, wherein the subject        is seronegative with respect to all dengue serotypes.        53. The method of any one of items 42 to 51, wherein the subject        is seropositive with respect to at least one dengue serotype.        54. The method of any one of items 42 to 53, wherein the        neutralizing antibody titers of the subject when tested at day        180 or day 365 after at least a first administration of said        reconstituted unit dose, and optionally a second administration        of said reconstituted unit dose 90 days after said first        administration, provide a ratio of not more than 50, or not more        than 40, or nor more than 30, or not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 4.        55. The method of item 54, wherein said neutralizing antibody        titers of the subject further provide a ratio of not more than        20 for the neutralizing antibody titer of dengue serotype 2 to        the neutralizing antibody titer of dengue serotype 1, and/or a        ratio of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        56. The method of any one of items 42 to 55, wherein the subject        is of 2 and 60 years of age.        57. The method of any one of items 42 to 55, wherein the subject        is under 9 years of age, under 4 years of age, or under 2 years        of age.        58. The method of any one of items 23 to 57, wherein the        reconstituted unit dose is obtained from the kit according to        item 20 or 21.        59. The reconstituted unit dose of any one of items 17 to 19 for        use in a method of items 23 to 58.        60. Use of a reconstituted unit dose of any one of items 17 to        19 for the manufacture of a medicament for a method according to        items 23 to 58.

Second List of Items of the Invention

1. A unit dose of a dengue vaccine composition comprising:a tetravalent dengue virus composition including four live, attenuateddengue virus strains wherein the unit dose is lyophilized and uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises:

-   -   (v) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (vi) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (vii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (viii) a chimeric dengue serotype 2/4 strain in a concentration        of at least 4.5 log 10 pfu/0.5 mL.        2. The unit dose of item 1, wherein upon reconstitution with 0.5        mL of a pharmaceutically acceptable diluent    -   (i) has a concentration of 3.3 log 10 pfu/0.5 mL to 5.0 log 10        pfu/0.5 mL,    -   (ii) has a concentration of 2.7 log 10 pfu/0.5 mL to 4.9 log 10        pfu/0.5 mL,    -   (iii) has a concentration of 4.0 log 10 pfu/0.5 mL to 5.7 log 10        pfu/0.5 mL, and    -   (iv) has a concentration of 4.5 log 10 pfu/0.5 mL to 6.2 log 10        pfu/0.5 mL.        3. The unit dose of item 1, wherein upon reconstitution with 0.5        mL of a pharmaceutically acceptable diluent:    -   (i) has a concentration of 3.3 log 10 pfu/dose to 5.0 log 10        pfu/0.5 mL,    -   (ii) has a concentration of 2.7 log 10 pfu/dose to 4.9 log 10        pfu/0.5 mL,    -   (iii) has a concentration of 4.0 log 10 pfu/dose to 5.7 log 10        pfu/0.5 mL, and    -   (iv) has a concentration of 4.5 log 10 pfu/dose to 5.5 log 10        pfu/0.5 mL.        4. The unit dose of any one of items 1 to 3, wherein upon        reconstitution with 0.5 mL of a pharmaceutically acceptable        diluent    -   (v) has a concentration of 3.3 log 10 pfu/0.5 mL to 3.6 log 10        pfu/0.5 mL,    -   (vi) has a concentration of 2.7 log 10 pfu/0.5 mL to 4.0 log 10        pfu/0.5 mL,    -   (vii) has a concentration of 4.0 log 10 pfu/0.5 mL to 4.6 log 10        pfu/0.5 mL, and    -   (viii) has a concentration of 4.5 log 10 pfu/0.5 mL to 5.1 log        10 pfu/0.5 mL.        5. The unit dose of any one of items 1 to 4, wherein upon        reconstitution with a pharmaceutically acceptable diluent (i),        (ii), (iii), and (iv) provide a total concentration of pfu/0.5        mL and based on said total concentration the concentration        of (ii) in pfu/0.5 mL is less than 10% or less than 8%, and the        concentration of (iv) in pfu/0.5 mL is at least 50%.        6. The unit dose of item 5, wherein upon reconstitution with a        pharmaceutically acceptable diluent (i), (ii), (iii), and (iv)        provide a total concentration of pfu/0.5 mL and based on said        total concentration the concentration of (i) in pfu/0.5 mL is at        least 1%, and the concentration of (iii) in pfu/0.5 mL is at        least 7% or at least 8%.        7. The unit dose of any one of items 1 to 6, wherein upon        reconstitution with a pharmaceutically acceptable diluent (i),        (ii), (iii), and (iv) provide a total concentration of pfu/0.5        mL and based on said total concentration the concentration        of (i) in pfu/0.5 mL is 1% to 7% of the total        concentration, (ii) in pfu/0.5 mL is less than 8% of the total        concentration, such as in the range of 1% to 8% of the total        concentration, (iiii) in pfu/0.5 mL is at least 10% of the total        concentration, and (iv) in pfu/0.5 mL is at least 65% of the        total concentration, such as in the range of 65% to 80%.        8. The unit dose of any one of items 1 to 7, wherein the        arithmetic sum of all four serotypes is in the range of 4.6 log        10 pfu/0.5 mL to 6.7 log 10 pfu/0.5 mL, preferably in the range        of 4.6 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL.        9. The unit dose of any one of items 1 to 8, wherein        reconstitution with a pharmaceutically acceptable diluent is        made with 0.5 ml of the pharmaceutically acceptable diluent.        10. The unit dose of any one of items 1 to 9, wherein the        lyophilized unit dose is prepared from a solution further        comprising a non-reducing sugar, a surfactant, a protein and an        inorganic salt.        11. The unit dose of item 10, wherein the non-reducing sugar is        trehalose, the surfactant is poloxamer 407, the protein is human        serum albumin and the inorganic salt is sodium chloride.        12. The unit dose of item 10 or 11, wherein the solution        comprises:    -   from about 10% (w/v) to about 20% (w/v) α,α-trehalose dihydrate        or an equimolar amount of other forms of α,α-trehalose,    -   from about 0.5% (w/v) to about 1.5% (w/v) poloxamer 407,    -   from about 0.05% (w/v) to about 2% (w/v) human serum albumin,        and    -   from about 70 mM to about 140 mM sodium chloride.        13. The unit dose of any one of items 1 to 12, wherein the        lyophilized unit dose is prepared from a solution comprising:    -   about 15% (w/v) α,α-trehalose dihydrate,    -   about 1% (w/v) poloxamer 407,    -   about 0.1% (w/v) human serum albumin, and    -   and about 100 mM sodium chloride.        14. The unit dose of any one of items 1 to 13, wherein each one        of the four live attenuated dengue virus strains has attenuating        mutations in the 5′-noncoding region (NCR) at nucleotide 57 from        cytosine to thymidine, in the NS1 gene at nucleotide 2579 from        guanine to adenine resulting in an amino acid change at position        828 from glycine to asparagine, and in the NS3 gene at        nucleotide 5270 from adenine to thymine resulting in an amino        acid change at position 1725 from glutamine to valine,        preferably further comprising one or more of the mutations        selected from the list comprising:    -   a) a mutation in the NS2A gene at nucleotide 4018 from cytosine        to thymidine resulting in an amino acid at position 1308 from        leucine to phenylalanine,    -   b) a silent mutation in the NS3 gene at nucleotide 5547 from        thymidine to cytosine, and    -   c) a mutation in the NS4A gene at nucleotide 6599 from guanine        to cytosine resulting in an amino acid change at position 2168        from glycine to alanine.        15. The unit dose of item 14, wherein (i) further comprises one        or more of the mutations selected from the list comprising:    -   a mutation in the NS2A gene at nucleotide 3823 from adenine to        cytosine resulting in an amino acid change at position 1243 from        isoleucine to leucine,    -   a mutation in the NS2B gene at nucleotide 4407 from adenine to        thymidine resulting in an amino acid change at position 1437        from glutamine to asparagine, and    -   a silent mutation in the NS4B gene at nucleotide 7311 from        adenine to guanine.        16. The unit dose of item 14 or 15, wherein (ii) further        comprises one or more of the mutations selected from the list        comprising:    -   a mutation in the prM gene at nucleotide 592 from adenine to        guanine resulting in an amino acid change at position 166 from        lysine to glutamine, and    -   a mutation in the NS5 gene at nucleotide 8803 from adenine to        guanine resulting in an amino acid change at position 2903 from        isoleucine to valine.        17. The unit dose of any one of items 14 to 16, wherein (iii)        further comprises one or more of the mutations selected from the        list comprising:    -   a mutation in the E gene at nucleotide 1603 from adenine to        thymidine resulting in an amino acid change at position 503 from        threonine to serine, and    -   a silent mutation in the NS5 gene at nucleotide 7620 from        adenine to guanine.        18. The unit dose of any one of items 14 to 17, wherein (iv)        further comprises one or more of the mutations selected from the        list comprising    -   a silent mutation in the C gene at nucleotide 225 from adenine        to thymidine,    -   a mutation in the NS2A gene at nucleotide 3674 from adenine to        guanine resulting in an amino acid change at position 1193 from        asparagine to glycine,    -   a mutation in the NS2A gene at nucleotide 3773 from adenine to        an adenine/guanine mix resulting in an amino acid change at        position 1226 from lysine to a lysine/asparagine mix,    -   a silent mutation in the NS3 gene at nucleotide 5391 from        cytosine to thymidine,    -   a mutation in the NS4A gene at nucleotide 6437 from cytosine to        thymidine resulting in an amino acid change at position 2114        from alanine to valine,    -   a silent mutation in the NS4B gene at nucleotide 7026 from        thymidine to a thymidine/cytosine mix, and    -   a silent mutation in the NS5 gene at nucleotide 9750 from        adenine to cytosine.        19. The unit dose of any one of items 1 to 18, wherein    -   (v) has the amino acid sequence of SEQ ID NO. 2,    -   (vi) has the amino acid sequence of SEQ ID NO. 4,    -   (vii) has the amino acid sequence of SEQ ID NO. 6, and    -   (viii) has the amino acid sequence of SEQ ID NO. 8.        20. The unit dose of any one of items 1 to 19 reconstituted with        0.3 to 0.8 mL of liquid for reconstitution.        21. The unit dose of item 20 reconstituted with 0.5 mL of liquid        for reconstitution.        22. The unit dose of item 20 or 21, wherein the liquid for        reconstitution is 37 mM aqueous sodium chloride solution.        23. A kit for preparing a reconstituted unit dose comprising the        following components:        a) a unit dose of any one of items 1 to 19, and        b) a pharmaceutically acceptable diluent for reconstitution.        24. The kit of item 23, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.        25. Container, such as a vial, comprising one to ten unit doses        of any one of items 1 to 22.        26. A method of preventing dengue disease in a subject        population comprising administering to the subject population a        reconstituted unit dose of any one of items 20 to 22.        27. A method of preventing virologically confirmable dengue        disease in a subject population comprising administering to the        subject population a reconstituted unit dose of a tetravalent        dengue virus composition including four live, attenuated dengue        virus strains.        28. A method of preventing virologically confirmable dengue        disease with hospitalization in a subject population comprising        administering to the subject population a reconstituted unit        dose of a tetravalent dengue virus composition including four        live, attenuated dengue virus strains.        29. The method of items 26 to 28, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of said unit dose 90 days after said first        administration, provide a ratio of not more than 50, or not more        than 40, or nor more than 30, or not more than 20 for the GMT of        dengue serotype 2 to the GMT of dengue serotype 4.        30. The method of item 29, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        31. A method of preventing dengue disease in a subject        comprising administering to the subject a reconstituted unit        dose of any one of items 20 to 22.        32. A method of preventing virologically confirmable dengue        disease in a subject comprising administering to the subject a        reconstituted unit dose of a tetravalent dengue virus        composition including four live, attenuated dengue virus        strains.        33. A method of preventing virologically confirmable dengue        disease with hospitalization in a subject comprising        administering to the subject a reconstituted unit dose of a        tetravalent dengue virus composition including four live,        attenuated dengue virus strains.        34. The method of items 31 to 33, wherein the neutralizing        antibody titers of the subject when tested at day 180 or day 365        after at least a first administration of said unit dose, and        optionally a second administration of said unit dose 90 days        after said first administration, provide a ratio of not more        than 50, or not more than 40, or nor more than 30, or not more        than 20 for the neutralizing antibody titer of dengue serotype 2        to the neutralizing antibody titer of dengue serotype 4.        35. The method of item 34, wherein said neutralizing antibody        titers of the subject further provide a ratio of not more than        20 for the neutralizing antibody titer of dengue serotype 2 to        the neutralizing antibody titer of dengue serotype 1, and/or a        ratio of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        36. The method of any one of items 26 to 35, wherein the method        is for preventing dengue hemorrhagic fever (DHF) or dengue shock        syndrome (DSS).        37. The method of any one of items 26 to 36, wherein the        reconstituted unit dose is administered by subcutaneous        injection, preferably to the deltoid region of the arm.        38. The method of any one of items 26 to 37, wherein two        reconstituted unit doses of any one of items 20 to 22 are        administered within 12 months or more.        39. The method of any one of items 26 to 37, wherein two        reconstituted unit doses of any one of items 20 to 22 are        administered within six months, preferably within three months.        40. The method of item 39, wherein the two reconstituted unit        doses are administered at day 0 and day 90 or at day 1 and day        90.        41. The method of item 38 to 40, wherein a third unit dose is        administered after administration of the second unit dose,        preferably within 12 months after administration of the first        unit dose.        42. The method of item 38 to 40, wherein a third unit dose is        administered after administration of the second unit dose,        preferably within 12 months after administration of the second        unit dose.        43. The method of any one of items 26 to 42, wherein the subject        or subject population is seronegative with respect to all dengue        serotypes.        44. The method of any one of items 26 to 42, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        45. The method of any one of items 26 to 44, wherein the subject        or subject population is of 2 to 60 years of age.        46. The method of any one of items 26 to 44, wherein the subject        or subject population is of 2 to 17 years of age.        47. The method of any one of items 26 to 44, wherein the subject        or subject population is under 9 years of age, under 4 years of        age, or under 2 years of age or from 2 to 9 years of age, or        from 2 to 5 years of age, or from 4 to 9 years of age or from 6        to 9 years of age, and optionally wherein the subject is        seronegative with respect to all dengue serotypes.        48. The method of any one of items 26 to 44, wherein the subject        or subject population is of 4 to 16 years of age.        49. The method of item 48, wherein the subject or subject        population is of 4 to 5 years of age 50. The method of item 48,        wherein the subject or subject population is of 6 to 11 years of        age.        51. The method of item 48, wherein the subject or subject        population is of 12 to 16 years of age.        52. The method of any one of items 26 to 51, wherein the subject        or subject population is from a dengue endemic region.        53. The method of any one of items 26 to 51, wherein the subject        or subject population is from a dengue non-endemic region.        54. The method of any one of items 52 or 53, wherein the subject        or subject population is from Asia Pacific or Latin America.        55. The method of any one of items 26 to 54, wherein the subject        or subject population has been subject to prior vaccination        against Yellow Fever, wherein prior vaccination against Yellow        Fever refers to a vaccination prior to the second administration        or prior to the first administration.        56. The method of any one of items 26 to 54, wherein the subject        or subject population is has been subject to prior vaccination        against Japanese Encephalitis, wherein prior vaccination against        Japanese Encephalitis refers to a vaccination prior to the        second administration or prior to the first administration.        57. The method of any one of items 26 to 54, wherein the subject        or subject population is has not been subject to prior        vaccination against Yellow Fever.        58. The method of any one of items 26 to 54, wherein the subject        or subject population is has not been subject to prior        vaccination against Japanese Encephalitis.        59. The method of any one of items 26 to 58 having a combined        vaccine efficacy against all four serotypes with a 2-sided 95%        confidence interval, wherein the lower bound is more than 25%,        when measured against placebo in a subject population of at        least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, about 30 days after the second        administration of the administration schedule until at least 12        months after the second administration of the administration        schedule.        60. The method of item 59, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, is more than 55%, is        more than 60%, is more than 65%, is more than 70% or is more        than 72%.        61. The method of any one of items 26 to 60 having a combined        vaccine efficacy against all four serotypes of more than 30%,        when measured against placebo in a subject population of at        least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, 30 days after the second administration        until at least 12 months after the second administration.        62. The method of item 61, wherein the combined vaccine efficacy        against all four serotypes is more than 40%, is more than 50%,        is more than 55%, is more than 60%, is more than 65%, is more        than 70%, is more than 75% is more than 78%, is more than 79% or        is about 80%.        63. The method of any one of items 26 to 62 having a combined        relative risk against all four serotypes with a 2-sided 95%        confidence interval, wherein the upper bound is less than 0.75,        when measured against placebo in a subject population of at        least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, 30 days after the second administration        until at least 12 months after the second administration.        64. The method of item 63, wherein the upper bound is less than        0.70, is less than 0.65, is less than 0.60, is less than 0.55,        is less than 0.50, is less than 0.45, is less than 0.40, is less        than 0.35, is less than 0.30 or is less than 0.28.        65. The method of any one of items 26 to 64, wherein the        combined relative risk against all four serotypes is less than        0.70, when measured against placebo in a subject population of        at least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, 30 days after the second administration        until at least 12 months after the second administration.        66. The method of item 65, wherein the combined relative risk        against all four serotypes is less than 0.65, is less than 0.60,        is less than 0.55, is less than 0.50, is less than 0.45, is less        than 0.40, is less than 0.35, is less than 0.30, is less than        0.25 or is less than 0.23.        67. The method of any one of items 26 to 58 having a combined        vaccine efficacy against all four serotypes with a 2-sided 95%        confidence interval, wherein the lower bound is more than 61.0%,        or more than 65.0% or more than 70.0% or more than 72.0% when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects from endemic irrespective of        serostatus at baseline and being selected from the group        consisting of 4 to 16 year old subjects at the time of        randomization, wherein said unit dose or said placebo is        administered at least twice within 6 months or less, about 30        days after the last administration of the administration        schedule until at least 12 or 13 months after the last        administration of the administration schedule.        68. The method of any one of items 26 to 58 having a combined        vaccine efficacy against all four serotypes of more than 66%, or        of more than 70%, or of more than 75%, or of more than 77%, or        of more than 80%, when measured against placebo in a subject        population of at least 5,000 healthy subjects, or at least        10,000 healthy subjects, or at least 15,000 healthy subjects        from endemic areas irrespective of serostatus at baseline and        being selected from the group consisting of 4 to 16 year old        subjects at the time of randomization, wherein said unit dose or        said placebo is administered at least twice within 6 months or        less, about 30 days after the last administration of the        administration schedule until at least 12 months or 13 month        after the last administration of the administration schedule.        69. The method of item 67 or 68, wherein the combined vaccine        efficacy against all four serotypes is measured about 30 days        after the last administration of the administration schedule        until 12 or 13 months after the last administration of the        administration schedule.        70. The method of item 67 or 68, wherein said unit dose or said        placebo is administered twice within three months, in particular        at about day 1 and about day 90, and wherein the combined        vaccine efficacy against all four serotypes is measured 30 days        after the second administration until 12 or 13 months after the        second administration of the administration schedule.        71. The method of any one of items 26 to 70 being effective and        safe.        72. The method of any one of items 26 to 71 having a relative        risk for virologically confirmed dengue with hospitalization        which is 1 or less, or 0.8 or less, or 0.6 or less, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects.        73. The method of any one of items 59 to 72, wherein the healthy        subjects of the subject population are of 4 to 16 years of age        at the time of randomization.        74. The method of any one of items 59 to 72, wherein the healthy        subjects of the subject population are of 4 to 5 years of age at        the time of randomization        75. The method of any one of items 59 to 72, wherein the healthy        subjects of the subject population are of 6 to 11 years of age        at the time of randomization.        76. The method of any one of items 59 to 72, wherein the healthy        subjects of the subject population are of 12 to 16 years of age        at the time of randomization.        77. The method of any one of items 59 to 72, wherein the healthy        subjects of the subject population are from Asia Pacific or        Latin America.        78. The method of any one of items 59 to 77, wherein the healthy        subjects of the subject population are seropositive with respect        to at least one serotype at baseline.        79. The method of any one of items 59 to 77, wherein the healthy        subjects of the subject population are seronegative with respect        to all serotypes at baseline.        80. The method of any one of items 59 to 79, wherein the healthy        subjects of the subject population have been subject to prior        vaccination against Yellow Fever.        81. The method of any one of items 59 to 79, wherein the healthy        subjects of the subject population have been subject to prior        vaccination against Japanese Encephalitis.        82. The method of any one of items 59 to 79, wherein the healthy        subjects of the subject population have not been subject to        prior vaccination against Yellow Fever.        83. The method of any one of items 59 to 79, wherein the healthy        subjects of the subject population have not been subject to        prior vaccination against Japanese Encephalitis.        84. A method for stimulating an immune response to all four        serotypes of dengue virus in a subject, comprising administering        to the subject a reconstituted unit dose of items 20 to 22.        85. The method of item 84, wherein the immune response to all        four serotypes of dengue virus is balanced.        86. The method of item 84 or 85, wherein the reconstituted unit        dose is administered by subcutaneous injection, preferably to        the deltoid region of the arm.        87. The method of any one of items 84 to 86, wherein two unit        doses of any one of items 20 to 22 are administered within 12        months or more.        88. The method of any one of items 84 to 87, wherein two        reconstituted unit doses of any one of items 20 to 22 are        administered within six months, preferably within three months.        89. The method of item 88, wherein the two reconstituted unit        doses are administered at day 0 and day 90 or at day 1 and day        90.        90. The method of item 87 to 89, wherein a third unit dose is        administered after the administration of the second unit dose,        preferably within 12 month of administration of the first unit        dose.        91. The method of item 87 to 89, wherein a third unit dose is        administered after the administration of the second unit dose,        preferably within 12 month of administration of the second unit        dose.        92. The method of any one of items 84 to 91, wherein the subject        is from a dengue endemic region.        93. The method of any one of items 84 to 91, wherein the subject        is from a dengue non-endemic region.        94. The method of any one of items 84 to 93, wherein the subject        is seronegative with respect to all dengue serotypes.        95. The method of any one of items 84 to 93, wherein the subject        is seropositive with respect to at least one dengue serotype.        96. The method of any one of items 84 to 95, wherein the        neutralizing antibody titers of the subject when tested at day        180 or day 365 after at least a first administration of said        reconstituted unit dose, and optionally a second administration        of said reconstituted unit dose 90 days after said first        administration, provide a ratio of not more than 50, or not more        than 40, or nor more than 30, or not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 4.        97. The method of item 96, wherein said neutralizing antibody        titers of the subject further provide a ratio of not more than        20 for the neutralizing antibody titer of dengue serotype 2 to        the neutralizing antibody titer of dengue serotype 1, and/or a        ratio of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        98. The method of any one of items 84 to 97, wherein the subject        is of 2 and 60 years of age.        99. The method of any one of items 84 to 97, wherein the subject        is under 9 years of age, under 4 years of age, or under 2 years        of age.        100. The method of any one of items 26 to 99, wherein the        reconstituted unit dose is obtained from the kit according to        item 23 or 24.        101. The reconstituted unit dose of any one of item 20 to 22 for        use in a method of items 26 to 100.        102. Use of a reconstituted unit dose of any one of items 20 to        22 for the manufacture of a medicament for a method according to        items 26 to 100.

Third List of Items of the Invention

1. A dengue vaccine composition for use in a method of preventingvirologically confirmable dengue disease in a subject comprisingconsecutively administering at least a first and a second unit dose ofthe dengue vaccine composition to the subject, wherein said first andsecond unit dose are administered subcutaneously within 3 months and atleast 4 weeks apart, optionally at about day 1 and at about day 90, andwherein the dengue vaccine composition is a tetravalent dengue viruscomposition including four live, attenuated dengue virus strainsrepresenting dengue serotype 1, dengue serotype 2, dengue serotype 3 anddengue serotype 4, wherein the attenuated dengue virus strains comprisechimeric dengue viruses and at least one non-chimeric dengue virus, andwherein the dengue serotype 1 and the dengue serotype 2 are present eachin a concentration based on the total concentration in pfu/0.5 mL whichis within 5%-points of each other and/or are together less than about10% of the total concentration in pfu/0.5 mL.2. The composition for use of item 1, wherein the method does notcomprise a determination of a previous dengue infection in the subjectbefore the administration of the first unit dose of the tetravalentdengue virus composition and wherein the method is safe and effective.3. The composition for use of item 1 or 2, wherein the dengue serotype 3is at least about 10% of the total concentration in pfu/0.5 mL and/orwherein the dengue serotype 4 is at least about 70% of the totalconcentration in pfu/0.5 mL.4. The composition for use of any one of items 1 to 3, wherein thedengue serotype 4 represents the highest concentration in thecomposition of all four serotypes, preferably with at least about 70% ofthe total concentration in pfu/0.5 mL, dengue serotype 3 represents thesecond highest concentration in the composition of all four serotypes,preferably with at least about 10% of the total concentration in pfu/0.5mL, and dengue serotype 1 and dengue serotype 2 each represent lowerconcentrations than the concentration of serotype 3, and optionallytogether represent less than about 10% of the total concentration inpfu/0.5 mL.5. The composition for use of any one of items 1 to 4, wherein thedengue serotype 1 is a chimeric dengue serotype 2/1 strain, the dengueserotype 2 is a non-chimeric dengue serotype 2 strain, the dengueserotype 3 is a chimeric dengue serotype 2/3 strain and the dengueserotype 4 is a chimeric dengue serotype 2/4 strain.6. The composition for use of any one of items 1 to 5, wherein thedengue serotype 1 has the amino acid sequence of SEQ ID NO. 2, thedengue serotype 2 has the amino acid sequence of SEQ ID NO. 4, thedengue serotype 3 has the amino acid sequence of SEQ ID NO. 6, and thedengue serotype 4 has the amino acid sequence of SEQ ID NO. 8.7. The composition for use of any one of items 1 to 6, wherein the unitdose upon reconstitution with 0.5 mL of a pharmaceutically acceptablediluent

-   -   (i) dengue serotype 1 has a concentration of 3.3 log 10 pfu/0.5        mL to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of 2.7 log 10 pfu/0.5        mL to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of 4.0 log 10        pfu/0.5 mL to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of 4.5 log 10 pfu/0.5        mL to 6.2 log 10 pfu/0.5 mL.        8. The composition for use of any one of items 1 to 7, wherein        the composition further comprises about 15% (w/v) α,α-trehalose        dihydrate, about 1% (w/v) poloxamer 407, about 0.1% (w/v) human        serum albumin, and about 100 mM sodium chloride when measured in        0.5 ml.        9. The composition for use of any one of items 1 to 8, wherein        the unit doses are administered to the deltoid region of the        arm.        10. The composition for use of any one of items 1 to 9, wherein        the subject is seronegative to all dengue serotypes at baseline        and/or is under 9 years of age.        11. The composition for use of any one of items 1 to 10, wherein        the subject is 4 to 5 years of age or 6 to 11 years of age or 12        to 16 years of age.        12. The composition for use of any one of items 1 to 11, wherein        the method is for preventing dengue hemorrhagic fever (DHF) or        dengue shock syndrome (DSS).        13. The composition for use of any one of items 1 to 12, wherein        the subject is from a dengue endemic region.        14. The composition for use of any one of items 1 to 12, wherein        the subject is from a dengue non-endemic region.        15. The composition for use of any one of items 1 or 14, wherein        the subject is from Asia Pacific or Latin America.        16. The composition for use of any one items of 1 to 15, wherein        the composition provides a seropositivity rate when it is        administered to a subject population of at least 50 subjects in        two unit doses subcutaneously at day 1 and at day 90, wherein        the subjects of the subject population are seronegative to all        dengue serotypes at baseline.        17. The composition for use of item 16, wherein at least one        month after administration of the first unit dose, such as at        day 30, at least 80% of the subject population are seropositive        for all four dengue serotypes.        18. The composition for use of item 16 or 17, wherein before or        at the time of the administration of the second unit dose, such        as at day 90, at least 80% of the subject population are        seropositive for all four dengue serotypes.        19. The composition for use of any one of items 16 to 18,        wherein after the administration of the second unit dose, such        as at day 120, at least 80%, or at least 85%, or at least 90% or        at least 95% of the subject population are seropositive for all        four dengue serotypes.        20. The composition for use of any one of items 11 to 14,        wherein after the administration of the second unit dose, such        as at day 270, at least 80%, or at least 85%, or at least 90% of        the subject population are seropositive for all four dengue        serotypes.        21. The composition for use of any one of items 16 to 20,        wherein the composition provides a seropositivity rate, when it        is administered to a subject population of at least 100 subjects        in two unit doses subcutaneously at day 1 and at day 90, wherein        the subjects of the subject population comprises from 20% to 40%        subjects who are seronegative to all dengue serotypes and from        60% to 80% subjects who are seropositive to at least one dengue        serotype at base line, wherein at day 120 and/or day 270 the        seropositivity rate for all four dengue serotypes in the        seronegative part of the subject population and the        seropositivity rate for all four dengue serotypes in the        seropositive part of the subject population do not deviate more        than 10%-points and/or wherein at day 120 the seropositivity        rate for all four dengue serotypes in the seronegative part of        the subject population and the seropositivity rate for all four        dengue serotypes in the seropositive part of the subject        population do not deviate more than 5%-points.        22. A method of inoculating a subject against virologically        confirmable dengue disease in a subject comprising administering        to the subject a tetravalent dengue virus composition including        four dengue virus strains representing serotype 1, serotype 2,        serotype 3 and serotype 4.        23. A method of inoculating a subject against virologically        confirmable dengue disease consisting of administering to the        subject a tetravalent dengue virus composition including four        dengue virus strains representing serotype 1, serotype 2,        serotype 3 and serotype 4.        24. The method of item 22 or 23, wherein the method does not        comprise a determination of a previous dengue infection in the        subject before the administration of the tetravalent dengue        virus composition.        25. The method of any one of items 22 to 24, wherein the        inoculation is safe irrespective of whether there is a        determination that the subject had a previous dengue infection        before the administration of the tetravalent dengue virus        composition.        26. The method of any one of items 22 to 25 which is safe.        27. The method of any one of items 22 to 26 which is effective.        28. The method of any one of items 22 to 27, wherein the virus        strains are live, attenuated dengue virus strains.        29. The method of any one of items 22 to 28, wherein the        composition includes at least one chimeric dengue virus and        optionally at least one non-chimeric dengue virus.        30. The method of any one of items 22 to 29, wherein the        composition includes a chimeric dengue serotype 2/1 strain and a        dengue serotype 2 strain and a chimeric dengue serotype 2/3        strain and a chimeric dengue serotype 2/4 strain.        31. The method of any one of items 22 to 30, wherein the subject        is seronegative to all dengue serotypes at base line and/or        under 9 years of age, 4 to 5 years of age, 6 to 11 years of age        or 12 to 16 years of age.        32. The method of any one of items 22 to 31, wherein the        composition is administered by subcutaneous injection.        33. The method of any one of items 22 to 32 including        consecutively administering at least a first and a second unit        dose of the dengue vaccine composition to the subject, wherein        said first and second unit dose are administered subcutaneously        within 3 months and at least four 4 apart, optionally at about        day 1 and at about day 90.        34. The method of any one of items 22 to 33, wherein the dengue        serotypes 1 and 2 are present in similar amounts and/or make up        less than about 10% of the total viral concentration.        35. The method of any one of items 22 to 34, wherein the dengue        serotype 3 makes up at least about 10% of the total viral        concentration.        36. The method of any one of items 22 to 35, wherein the dengue        serotype 4 makes up at least about 70% of the total viral        concentration.        37. The method of any one of items 22 to 36 wherein the dengue        serotype 4 represents the highest concentration in the        composition of all four serotypes, preferably with at least        about 70% of the total concentration in pfu/0.5 mL, dengue        serotype 3 represents the second highest concentration in the        composition of all four serotypes, preferably with at least        about 10% of the total concentration in pfu/0.5 mL, and dengue        serotype 1 and dengue serotype 2 each represent lower        concentrations than the concentration of serotype 3, and        optionally together represent less than about 10% of the total        concentration in pfu/0.5 mL.        38. The method of any one of items 22 to 37, wherein the method        is for preventing dengue hemorrhagic fever (DHF) or dengue shock        syndrome (DSS).        39. The method of any one of items 22 to 38 wherein the subject        or subject population is from a dengue endemic region.        40. The method of any one of items 22 to 38, wherein the subject        or subject population is from a dengue non-endemic region.        41. The method of any one of items 22 to 40, wherein the subject        or subject population is from Asia Pacific or Latin America.        42. The method of any one of items 22 to 41 having a combined        vaccine efficacy against all four dengue serotypes with a        2-sided 95% confidence interval, wherein the lower bound is more        than 25%, when measured against placebo in a subject population        of at least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, and optionally at least 4 weeks apart, about        30 days after the second administration of the administration        schedule until at least 12 months after the second        administration of the administration schedule.        43. The method of item 42, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, is more than 55%, is        more than 60%, is more than 65%, is more than 70% or is more        than 72%.        44. The method of any one of items 22 to 43 having a combined        vaccine efficacy against all four dengue serotypes of more than        30%, when measured against placebo in a subject population of at        least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, and optionally at least 4 weeks apart, 30        days after the second administration until at least 12 months        after the second administration.        45. The method of item 44, wherein the combined vaccine efficacy        against all four dengue serotypes is more than 40%, is more than        50%, is more than 55%, is more than 60%, is more than 65%, is        more than 70%, is more than 75% is more than 78%, is more than        79% or is about 80%.        46. The method of any one of items 22 to 45 having a combined        vaccine efficacy against all four dengue serotypes in        seronegative subjects with a 2-sided 95% confidence interval,        wherein the lower bound is more than 25%, when measured against        placebo in a subject population of at least 1,500 or at least        2,000 healthy subjects being seronegative against all serotypes        at baseline, wherein said unit dose or said placebo is        administered at least twice within less than 6 months, such as        within 3 months, about 30 days after the second administration        of the administration schedule until at least 12 months after        the second administration of the administration schedule.        47. The method of item 46, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, or is more than 55%.        48. The method of any one of items 22 to 47 having a combined        vaccine efficacy against all four dengue serotypes in        seronegative subjects of more than 30%, when measured against        placebo in a subject population of at least 1,500 or at least        2,000 healthy subjects being seronegative against all serotypes        at baseline, wherein said unit dose or said placebo is        administered at least twice within less than 6 months, such as        within 3 months, 30 days after the second administration until        at least 12 months after the second administration.        49. The method of item 48, wherein the combined vaccine efficacy        against all four dengue serotypes in seronegative subjects is        more than 40%, is more than 50%, is more than 60%, is more than        65%, or is more than 70%.        50. The method of any one of items 22 to 49 having a combined        vaccine efficacy against all four dengue serotypes with a        2-sided 95% confidence interval, wherein the lower bound is more        than 25%, when measured against placebo in a subject population        of at least 1,000 healthy subjects 4 to 5 years of age at the        time of randomization and irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, about 30 days after the second administration of the        administration schedule until at least 12 months after the        second administration of the administration schedule.        51. The method of item 50, wherein the lower bound is more than        30%, is more than 40%, is more than 45%.        52. The method of any one of items 22 to 51 having a combined        vaccine efficacy against all four dengue serotypes of more than        30%, when measured against placebo in a subject population of at        least 1,000 healthy subjects 4 to 5 years of age at the time of        randomization and irrespective of serostatus at baseline,        wherein said unit dose or said placebo is administered at least        twice within less than 6 months, such as within 3 months, 30        days after the second administration until at least 12 months        after the second administration.        53. The method of item 52, wherein the combined vaccine efficacy        against all four dengue serotypes is more than 40%, is more than        50%, is more than 60%, is more than 65%, or is more than 70%.        54. The method of any one of items 22 to 53 having a combined        vaccine efficacy against all four dengue serotypes with a        2-sided 95% confidence interval, wherein the lower bound is more        than 25%, when measured against placebo in a subject population        of at least 1,000 healthy subjects 6 to 11 years of age at the        time of randomization and irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, about 30 days after the second administration of the        administration schedule until at least 12 months after the        second administration of the administration schedule.        55. The method of item 54, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, is more than 60%, or is        more than 70%.        56. The method of any one of items 22 to 55 having a combined        vaccine efficacy against all four dengue serotypes of more than        30%, when measured against placebo in a subject population of at        least 1,000 healthy subjects 6 to 11 years of age at the time of        randomization and irrespective of serostatus at baseline,        wherein said unit dose or said placebo is administered at least        twice within less than 6 months, such as within 3 months, 30        days after the second administration until at least 12 months        after the second administration.        57. The method of item 56, wherein the combined vaccine efficacy        against all four dengue serotypes is more than 40%, is more than        50%, is more than 60%, is more than 70%, is more than 75%, or is        more than 80%.        58. The method of any one of items 26 to 57 having a combined        vaccine efficacy against all four dengue serotypes with a        2-sided 95% confidence interval, wherein the lower bound is more        than 25%, when measured against placebo in a subject population        of at least 1,000 healthy subjects 12 to 16 years of age at the        time of randomization and irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, about 30 days after the second administration of the        administration schedule until at least 12 months after the        second administration of the administration schedule.        59. The method of item 58, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, is more than 60%, is        more than 65%, or is more than 68%.        60. The method of any one of items 26 to 59 having a combined        vaccine efficacy against all four dengue serotypes of more than        30%, when measured against placebo in a subject population of at        least 1,000 healthy subjects 12 to 16 years of age at the time        of randomization and irrespective of serostatus at baseline,        wherein said unit dose or said placebo is administered at least        twice within less than 6 months, such as within 3 months, 30        days after the second administration until at least 12 months        after the second administration.        61. The method of item 60, wherein the combined vaccine efficacy        against all four dengue serotypes is more than 40%, is more than        50%, is more than 60%, is more than 70%, is more than 75%, or is        more than 80%.        62. The method of any one of items 22 to 61 having a vaccine        efficacy against dengue serotype 1 with a 2-sided 95% confidence        interval, wherein the lower bound is more than 25%, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, about 30 days after the second administration of the        administration schedule until at least 12 months after the        second administration of the administration schedule.        63. The method of item 62, wherein the lower bound is more than        30%, is more than 40%, or is more than 50%.        64. The method of any one of items 22 to 63 having a vaccine        efficacy against dengue serotype 1 of more than 30%, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, 30 days after the second administration until at least        12 months after the second administration.        65. The method of item 64, wherein the vaccine efficacy against        dengue serotype 1 is more than 40%, is more than 50%, is more        than 60%, is more than 65%, or is more than 70%.        66. The method of any one of items 22 to 65 having a vaccine        efficacy against dengue serotype 2 with a 2-sided 95% confidence        interval, wherein the lower bound is more than 25%, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, about 30 days after the second administration of the        administration schedule until at least 12 months after the        second administration of the administration schedule.        67. The method of item 66, wherein the lower bound is more than        30%, is more than 40%, is more than 50, is more than 60, is more        than 70, is more than 80, or is more than 90%.        68. The method of any one of items 22 to 67 having a vaccine        efficacy against dengue serotype 2 of more than 30%, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, 30 days after the second administration until at least        12 months after the second administration.        69. The method of item 68, wherein the vaccine efficacy against        dengue serotype 2 is more than 40%, is more than 50%, is more        than 60%, is more than 70%, is more than 80, or is more than        90%.        70. The method of any one of items 22 to 69 having a vaccine        efficacy against dengue serotype 3 with a 2-sided 95% confidence        interval, wherein the lower bound is more than 25%, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, about 30 days after the second administration of the        administration schedule until at least 12 months after the        second administration of the administration schedule.        71. The method of item 70, wherein the lower bound is more than        30%, is more than 40%.        72. The method of any one of items 22 to 71 having a vaccine        efficacy against dengue serotype 3 of more than 30%, when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects irrespective of serostatus at        baseline, wherein said unit dose or said placebo is administered        at least twice within less than 6 months, such as within 3        months, 30 days after the second administration until at least        12 months after the second administration.        73. The method of item 72, wherein the vaccine efficacy against        dengue serotype 3 is more than 40%, is more than 50%, is more        than 55%, or is more than 60%.        74. The method of any one of items 22 to 73 having a combined        vaccine efficacy against virologically-confirmed dengue with        hospitalization against all four serotypes with a 2-sided 95%        confidence interval, wherein the lower bound is more than 25%,        when measured against placebo in a subject population of at        least 1,500 or at least 2,000 healthy subjects being        seronegative against all serotypes at baseline, wherein said        unit dose or said placebo is administered at least twice within        less than 6 months, such as within 3 months, about 30 days after        the second administration of the administration schedule until        at least 12 months after the second administration of the        administration schedule.        75. The method of item 74, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, is more than 60%, is        more than 70%, or is more than 75%.        76. The method of any one of items 22 to 75 having a combined        vaccine efficacy against virologically-confirmed dengue with        hospitalization against all four serotypes of more than 30%,        when measured against placebo in a subject population of at        least 1,500 or at least 2,000 healthy subjects, healthy subjects        being seronegative against all serotypes at baseline, wherein        said unit dose or said placebo is administered at least twice        within less than 6 months, such as within 3 months, 30 days        after the second administration until at least 12 months after        the second administration.        77. The method of item 76, wherein the combined vaccine efficacy        against virologically-confirmed dengue with hospitalization        against all four serotypes is more than 40%, is more than 50%,        is more than 60%, is more than 70%, is more than 80%, or is more        than 90%.        78. The method of any one of items 21 to 77 having a combined        vaccine efficacy against virologically-confirmed dengue with        hospitalization against all four serotypes with a 2-sided 95%        confidence interval, wherein the lower bound is more than 25%,        when measured against placebo in a subject population of at        least 1,500 or at least 2,000 healthy subjects being        seropositive at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, about 30 days after the second        administration of the administration schedule until at least 12        months after the second administration of the administration        schedule.        79. The method of item 78, wherein the lower bound is more than        30%, is more than 40%, is more than 50%, is more than 60%, is        more than 70%, or is more than 80%.        80. The method of any one of items 22 to 79 having a combined        vaccine efficacy against virologically-confirmed dengue with        hospitalization against all four serotypes of more than 30%,        when measured against placebo in a subject population of at        least 1,500 or at least 2,000 healthy subjects, healthy subjects        being seropositive at baseline, wherein said unit dose or said        placebo is administered at least twice within less than 6        months, such as within 3 months, 30 days after the second        administration until at least 12 months after the second        administration.        81. The method of item 80, combined vaccine efficacy against        virologically-confirmed dengue with hospitalization against all        four serotypes is more than 40%, is more than 50%, is more than        60%, is more than 70%, is more than 80%, or is more than 90%.        82. The method of any one of items 22 to 81 having a combined        relative risk against all four dengue serotypes with a 2-sided        95% confidence interval, wherein the upper bound is less than        0.75, when measured against placebo in a subject population of        at least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, 30 days after the second administration        until at least 12 months after the second administration.        83. The method of item 82, wherein the upper bound is less than        0.70, is less than 0.65, is less than 0.60, is less than 0.55,        is less than 0.50, is less than 0.45, is less than 0.40, is less        than 0.35, is less than 0.30 or is less than 0.28.        84. The method of any one of items 22 to 83, wherein the        combined relative risk against all four dengue serotypes is less        than 0.70, when measured against placebo in a subject population        of at least 5,000 healthy subjects, or at least 10,000 healthy        subjects, or at least 15,000 healthy subjects irrespective of        serostatus at baseline, wherein said unit dose or said placebo        is administered at least twice within less than 6 months, such        as within 3 months, 30 days after the second administration        until at least 12 months after the second administration.        85. The method of item 84, wherein the combined relative risk        against all four serotypes is less than 0.65, is less than 0.60,        is less than 0.55, is less than 0.50, is less than 0.45, is less        than 0.40, is less than 0.35, is less than 0.30, is less than        0.25 or is less than 0.23.        86. The method of any one of items 22 to 85 having a combined        vaccine efficacy against all four serotypes with a 2-sided 95%        confidence interval, wherein the lower bound is more than 61.0%,        or more than 65.0 or more than 70.0% or more than 72.0% when        measured against placebo in a subject population of at least        5,000 healthy subjects, or at least 10,000 healthy subjects, or        at least 15,000 healthy subjects from endemic regions        irrespective of serostatus at baseline and being selected from        the group consisting of 4 to 16 year old subjects at the time of        randomization, wherein said unit dose or said placebo is        administered at least twice within 6 months or less, about 30        days after the last administration of the administration        schedule until at least 12 or 13 months after the last        administration of the administration schedule.        87. The method of any one of items 22 to 86 having a combined        vaccine efficacy against all four serotypes of more than 66%, or        of more than 70%, or of more than 75%, or of more than 77%, or        of more than 80.0%, when measured against placebo in a subject        population of at least 5,000 healthy subjects, or at least        10,000 healthy subjects, or at least 15,000 healthy subjects        from endemic regions irrespective of serostatus at baseline and        being selected from the group consisting of 4 to 16 year old        subjects at the time of randomization, wherein said unit dose or        said placebo is administered at least twice within 6 months or        less, about 30 days after the last administration of the        administration schedule until at least 12 months or 13 month        after the last administration of the administration schedule.        88. The method of any one of items 22 to 87, wherein said unit        dose or said placebo is administered at day 1 and day 90.        89. The method of any one of items 22 to 88 having a relative        risk for virologically confirmed dengue with hospitalization        which is 1 or less, or 0.8 or less, or 0.6 or less, when        measured against placebo in a subject population of at least        1,000 healthy subjects, or at least 5,000 healthy subjects, or        at least 10,000 healthy subjects irrespective of serostatus at        baseline and in age groups from 4 to 16 years, in particular in        subjects 4 to 5 years of age at the time of randomization.        90. The method of any one of items 22 to 89, wherein the        occurrence of vaccine related serious adverse events is less        than 0.1%.        91. The method of any one of items 22 to 90, wherein the        occurrence of vaccine related unsolicited adverse events        occurring within 4 weeks of administration is less than 2%.        92. The method of any one of items 22 to 91, wherein the        occurrence of vaccine related solicited adverse events occurring        within 2 weeks of administration is less than 35%.        93. The method of any one of items 22 to 92, wherein the        occurrence of solicited local reactions occurring within 1 weeks        of administration is less than 40%.        94. The method of any one of items 22 to 93, wherein the unit        dose upon reconstitution with 0.5 mL of a pharmaceutically        acceptable diluent    -   (i) dengue serotype 1 has a concentration of 3.3 log 10 pfu/0.5        mL to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of 2.7 log 10 pfu/0.5        mL to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of 4.0 log 10        pfu/0.5 mL to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of 4.5 log 10 pfu/0.5        mL to 6.2 log 10 pfu/0.5 mL, and optionally comprises about 15%        (w/v) α,α-trehalose dihydrate, about 1% (w/v) poloxamer 407,        about 0.1% (w/v) human serum albumin, and about 100 mM sodium        chloride when measured in 0.5 mL.        95. A reconstituted unit dose of a dengue vaccine composition        for use in a method of preventing virologically confirmable        dengue disease in a subject comprising consecutively        administering at least a first and a second unit dose of the        dengue vaccine composition to the subject, wherein said first        and second unit dose are administered subcutaneously within 3        months and at least 4 weeks apart, optionally at about day 1 and        at about day 90, wherein the dengue vaccine composition is a        tetravalent dengue virus composition including four dengue virus        strains representing dengue serotype 1, dengue serotype 2,        dengue serotype 3 and dengue serotype 4, optionally wherein the        dengue virus strains are live, attenuated, and wherein upon        reconstitution with 0.5 mL of a pharmaceutically acceptable        diluent    -   (i) dengue serotype 1 has a concentration of at least 3.3 log 10        pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of at least 2.7 log        10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of at least 4.0 log        10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of at least 4.5 log        10 pfu/0.5 mL.        96. The unit dose for use of item 95, wherein the subject is        under 9 years of age and/or when the serostatus of the subject        is unknown or seronegative.        97. The unit dose for use of item 95 or 96, which is effective.        98. The unit dose for use of any one of items 95 to 97, which is        effective against all four dengue serotypes.        99. The unit dose for use of any one of items 95 to 98, which is        safe.        100. The unit dose for use of any one of items 95 to 99, wherein        the unit dose includes at least one chimeric dengue virus.        101. The unit dose for use of any one of items 95 to 100,        wherein the unit dose includes at least one non-chimeric dengue        virus and at least one chimeric dengue virus.        102. The unit dose for use of any one of items 95 to 101,        wherein the subject is seronegative to all dengue serotypes at        baseline and/or is under 9 years of age.        103. The unit dose for use of any one of items 95 to 102,        wherein the subject is 4 to 5 years of age or 6 to 11 years of        age or 12 to 16 years of age.        104. The unit dose for use of any one of items 95 to 103,        wherein the method does not comprise a determination of a        previous dengue infection in the subject before the        administration of the first unit dose of the tetravalent dengue        virus composition.        105. The unit dose for use of any one of items 95 to 104,        wherein the dengue serotype 4 represents the highest        concentration in the composition of all four serotypes,        optionally with at least about 70% of the total concentration in        pfu/0.5 mL, dengue serotype 3 represents the second highest        concentration in the composition of all four serotypes with at        least about 10% of the total concentration in pfu/0.5 mL, and        dengue serotype 1 and dengue serotype 2 each represent lower        concentrations than the concentration of serotype 3 and together        represent less than about 10% of the total concentration in        pfu/0.5 mL and/or which are within 5%-points of each other.        106. The unit dose for use of any one of items 95 to 105,        wherein the dengue serotype 1 is a chimeric dengue serotype 2/1        strain, the dengue serotype 2 is a non-chimeric dengue serotype        2 strain, the dengue serotype 3 is a chimeric dengue serotype        2/3 strain and the dengue serotype 4 is a chimeric dengue        serotype 2/4 strain.        107. The unit dose for use of any one of items 95 to 106,        wherein the dengue serotype 1 has the amino acid sequence of SEQ        ID NO. 2, the dengue serotype 2 has the amino acid sequence of        SEQ ID NO. 4, the dengue serotype 3 has the amino acid sequence        of SEQ ID NO. 6, and the dengue serotype 4 has the amino acid        sequence of SEQ ID NO. 8.        108. The unit dose for use of any one of items 95 to 107,        wherein the unit dose further comprises from about 10% w/v to        about 20% w/v α,α-trehalose dihydrate or an equimolar amount of        other forms of α,α-trehalose, from about 0.5% w/v to about 1.5%        w/v poloxamer 407, from about 0.05% w/v to about 2% w/v human        serum albumin, and from about 70 mM to 140 mM sodium chloride        when measured in 0.5 mL.        109. The unit dose for use of any one of items 95 to 108,        wherein the unit dose further comprises about 15% (w/v)        α,α-trehalose dihydrate, about 1% (w/v) poloxamer 407, about        0.1% (w/v) human serum albumin, and about 100 mM sodium chloride        when measured in 0.5 mL.        110. The unit dose for use of any one of items 95 to 109,        wherein the method is for preventing dengue hemorrhagic fever        (DHF) or dengue shock syndrome (DSS).        111. The unit dose for use of any one of items 95 to 110,        wherein the subject is from a dengue endemic region.        112. The unit dose for use of any one of items 95 to 111,        wherein the subject is from a dengue non-endemic region.        113. The unit dose for use of any one of items 95 to 112,        wherein the subject is from Asia Pacific or Latin America.        114. The unit dose for use of any one items of 95 to 113,        wherein the unit dose provides a seropositivity rate when it is        administered to a subject population of at least 50 subjects in        two unit doses subcutaneously at day 1 and at day 90, wherein        the subjects of the subject population are seronegative to all        dengue serotypes at baseline.        115. The unit dose for use of item 114, wherein at least one        month after administration of the first unit dose, such as at        day 30, at least 80% of the subject population are seropositive        for all four dengue serotypes.        116. The unit dose for use of item 114 or 115, wherein before or        at the time of the administration of the second unit dose, such        as at day 90, at least 80% of the subject population are        seropositive for all four dengue serotypes.        117. The unit dose for use of any one of items 114 to 116,        wherein after the administration of the second unit dose, such        as at day 120, at least 80%, or at least 85%, or at least 90% or        at least 95% of the subject population are seropositive for all        four dengue serotypes.        118. The unit dose for use of any one of items 114 to 117,        wherein after the administration of the second unit dose, such        as at day 270, at least 80%, or at least 85%, or at least 90% of        the subject population are seropositive for all four dengue        serotypes.        119. The unit dose for use of any one of items 114 to 118,        wherein the unit dose provides a seropositivity rate, when it is        administered to a subject population of at least 100 subjects in        two unit doses subcutaneously at day 1 and at day 90, wherein        the subjects of the subject population comprises from 20% to 40%        subjects who are seronegative to all dengue serotypes and from        60% to 80% subjects who are seropositive to at least one dengue        serotype at base line, wherein at day 120 and/or day 270 the        seropositivity rate for all four dengue serotypes in the        seronegative part of the subject population and the        seropositivity rate for all four dengue serotypes in the        seropositive part of the subject population do not deviate more        than 10%-points and/or wherein at day 120 the seropositivity        rate for all four dengue serotypes in the seronegative part of        the subject population and the seropositivity rate for all four        dengue serotypes in the seropositive part of the subject        population do not deviate more than 5%-points.        120. The unit dose for use of any one of items 95 to 119,        wherein upon reconstitution with 0.5 mL of a pharmaceutically        acceptable diluent    -   (i) dengue serotype 1 has a concentration of 3.3 log 10 pfu/0.5        mL to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of 2.7 log 10 pfu/0.5        mL to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of 4.0 log 10        pfu/0.5 mL to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of 4.5 log 10 pfu/0.5        mL to 6.2 log 10 pfu/0.5 mL.        121. The unit dose for use of any one of items 95 to 120,        wherein upon reconstitution with 0.5 mL of a pharmaceutically        acceptable diluent:    -   (i) dengue serotype 1 has a concentration of 3.3 log 10 pfu/dose        to 5.0 log 10 pfu/0.5 mL,    -   (ii) dengue serotype 2 has a concentration of 2.7 log 10        pfu/dose to 4.9 log 10 pfu/0.5 mL,    -   (iii) dengue serotype 3 has a concentration of 4.0 log 10        pfu/dose to 5.7 log 10 pfu/0.5 mL, and    -   (iv) dengue serotype 4 has a concentration of 4.5 log 10        pfu/dose to 5.5 log 10 pfu/0.5 mL.

Fourth List of Items of the Invention

1. A yellow fever vaccine, in particular YF-17D, and a reconstitutedunit dose of a dengue vaccine composition for use in a method ofpreventing yellow fever and dengue disease in a subject population,wherein said reconstituted unit dose comprises a tetravalent denguevirus composition including four live, attenuated dengue virus strainsand upon reconstitution with 0.5 mL of a pharmaceutically acceptablediluent comprises

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        2. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 1, wherein        the geometric mean neutralizing antibody titers (GMTs) of the        subject population when tested in at least 40, or at least 50,        or at least 60 subjects at day 180 or day 365 after at least a        first administration of said unit dose, and optionally a second        administration of said reconstituted unit dose 90 days after        said first administration, provide a ratio of not more than 50,        or not more than 40, or not more than 30, or not more than 20        for the GMT of dengue serotype 2 to the GMT of dengue serotype        4.        3. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 2, wherein        said GMTs of the subject population further provide a ratio of        not more than 20 for the GMT of dengue serotype 2 to the GMT of        dengue serotype 1, and/or a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 3.        4. A yellow fever vaccine, in particular YF-17D, and a        reconstituted unit dose of a dengue vaccine composition for use        in a method of preventing yellow fever and dengue disease in a        subject, wherein said reconstituted unit dose comprises a        tetravalent dengue virus composition including four live,        attenuated dengue virus strains and upon reconstitution with 0.5        mL of a pharmaceutically acceptable diluent comprises    -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        5. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 4, wherein        the neutralizing antibody titers of the subject when tested at        day 180 or day 365 after at least a first administration of said        unit dose, and optionally a second administration of said unit        dose 90 days after said first administration, provide a ratio of        not more than 50, or not more than 40, or not more than 30, or        not more than 20 for the neutralizing antibody titer of dengue        serotype 2 to the neutralizing antibody titer of dengue serotype        4.        6. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 5, wherein        said neutralizing antibody titers of the subject further provide        a ratio not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 1, and/or a ratio of not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 3.        7. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 6, wherein the subject population or subject is        seronegative to all dengue serotypes.        8. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 7, wherein the administration of said reconstituted unit        dose and said yellow fever vaccine is simultaneous or        sequential.        9. The yellow fever vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 8, wherein said reconstituted unit dose is administered by        subcutaneous injection and said yellow fever vaccine is        administered by subcutaneous injection, wherein said injections        are preferably administered to the arm, more preferably to the        deltoid region of the arm.        10. The yellow fever vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to item 9,        wherein said reconstituted unit dose and said yellow fever        vaccine are administered to different anatomical sites, such as        to opposite arms.        11. The yellow fever vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 10, wherein two of said reconstituted unit doses are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        12. The yellow fever vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 11, wherein the method comprises the administration        of two of said reconstituted unit doses and one dose of said        yellow fever vaccine, in particular according to the following        schedule    -   an administration of said yellow fever vaccine on day 0,    -   a first administration of the first reconstituted unit dose        after said yellow fever vaccine administration, such as 3 months        later and preferably on day 90, and    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 180.        13. The yellow fever vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 11, wherein the method comprises the administration        of two of said reconstituted unit doses and one dose of said        yellow fever vaccine, in particular according to the following        schedule    -   a first administration of the first reconstituted unit dose on        day 0,    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 90, and    -   an administration of said yellow fever vaccine after said second        administration of the reconstituted unit dose, such as 3 months        later and preferably on day 180.        14. The yellow fever vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 11, wherein the method comprises the administration        of two of said reconstituted unit doses and one dose of said        yellow fever vaccine, in particular according to the following        schedule    -   a simultaneous administration of the first reconstituted unit        dose and said yellow fever vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said simultaneous administration, such as 3 months later        and preferably on day 90.        15. The yellow fever vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 14, wherein the subject population or subject is from        a dengue endemic region.        16. A method of preventing dengue disease in a subject,        comprising administering to the subject a reconstituted unit        dose of any one of items 1 to 15, wherein the method further        comprises preventing yellow fever in the subject by concomitant        administration of a yellow fever vaccine, in particular YF-17D,        to the subject.        17. The method of item 16, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of a reconstituted unit dose of any one of        items 17 to 19 90 days after said first administration, provide        a ratio of not more than 50, or not more than 40, or not more        than 30, or not more than 20 for the neutralizing antibody titer        of dengue serotype 2 to the neutralizing antibody titer of        dengue serotype 4.        18. The method of item 17, wherein said neutralizing antibody        titers of the subject further provide a ratio not more than 20        for the neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 1, and/or a ratio        of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        19. The method of any one of items 16 to 18, wherein the subject        population or subject is seronegative with respect to all dengue        serotypes.        20. The method of any one of items 16 to 19, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        21. The method of any one of items 16 to 20, wherein the        administration of said reconstituted unit dose and said yellow        fever vaccine is simultaneous or sequential.        22. The method of any one of items 16 to 22, wherein said        reconstituted unit dose is administered by subcutaneous        injection and said yellow fever vaccine is administered by        subcutaneous injection, wherein said injections are preferably        administered to the arm, more preferably to the deltoid region        of the arm.        23. The method of item 22, wherein said reconstituted unit dose        and said yellow fever vaccine are administered to different        anatomical sites, such as to opposite arms.        24. The method of any one of items 16 to 23, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        25. The method of any one of items 16 to 24, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 15 and one dose of a yellow fever vaccine, in        particular YF-17D, in particular according to the following        schedule    -   an administration of said yellow fever vaccine on day 0,    -   a first administration of the first reconstituted unit dose        after said yellow fever vaccine administration, such as 3 months        later and preferably on day 90, and    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 180.        26. The method of any one of items 16 to 25, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 15 and one dose of a yellow fever vaccine, in        particular YF-17D, in particular according to the following        schedule    -   a first administration of the first reconstituted unit dose on        day 0,    -   a second administration of the second reconstituted unit dose        after said first administration of the reconstituted unit dose,        such as 3 months later and preferably on day 90, and    -   an administration of said yellow fever vaccine after said second        administration of the reconstituted unit dose, such as 3 months        later and preferably on day 180.        27. The method of any one of items 16 to 27, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 15 and one dose of a yellow fever vaccine, in        particular YF-17D, in particular according to the following        schedule    -   a simultaneous administration of the first reconstituted unit        dose and said yellow fever vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said simultaneous administration, such as 3 months later        and preferably on day 90.        28. The method of any one of items 16 to 27, wherein the subject        population or subject is of 2 to 60 years of age.        29. The method of any one of items 16 to 28, wherein the subject        population or subject is from a dengue endemic region.        30. The method of any one of items 16 to 29, wherein the subject        population or subject is from a dengue non-endemic region,        preferably from a dengue non-endemic region and a yellow fever        non-endemic region.        31. A kit for comprising the following components:        a) a reconstituted unit dose of any one of items 1 to 15,        b) a pharmaceutically acceptable diluent for reconstitution of        said unit dose, and        c) a yellow fever vaccine.        32. The kit of item 31, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.

Fifth List of Items of the Present Invention

1. A hepatitis A vaccine, such as HAVRIX® or VAQTA®, and a reconstitutedunit dose of a dengue vaccine composition for use in a method ofpreventing hepatitis A and dengue disease in a subject population,wherein said reconstituted unit dose comprises a tetravalent denguevirus composition including four live, attenuated dengue virus strainsand upon reconstitution with 0.5 mL of a pharmaceutically acceptablediluent comprises

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        2. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 1, wherein        the geometric mean neutralizing antibody titers (GMTs) of the        subject population when tested in at least 40, or at least 50,        or at least 60 subjects at day 180 or day 365 after at least a        first administration of said unit dose, and optionally a second        administration of said reconstituted unit dose 90 days after        said first administration, provide a ratio of not more than 50,        or not more than 40, or not more than 30, or not more than 20        for the GMT of dengue serotype 2 to the GMT of dengue serotype        4.        3. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 2, wherein        said GMTs of the subject population further provide a ratio of        not more than 20 for the GMT of dengue serotype 2 to the GMT of        dengue serotype 1, and/or a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 3.        4. A hepatitis A vaccine, such as HAVRIX® or VAQTA®, and a        reconstituted unit dose of a dengue vaccine composition for use        in a method of preventing hepatitis A and dengue disease in a        subject, wherein said reconstituted unit dose comprises a        tetravalent dengue virus composition including four live,        attenuated dengue virus strains and upon reconstitution with 0.5        mL of a pharmaceutically acceptable diluent comprises    -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        5. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 4, wherein        the neutralizing antibody titers of the subject when tested at        day 180 or day 365 after at least a first administration of said        unit dose, and optionally a second administration of said unit        dose 90 days after said first administration, provide a ratio of        not more than 50, or not more than 40, or not more than 30, or        not more than 20 for the neutralizing antibody titer of dengue        serotype 2 to the neutralizing antibody titer of dengue serotype        4.        6. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 5, wherein        said neutralizing antibody titers of the subject further provide        a ratio not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 1, and/or a ratio of not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 3.        7. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 6, wherein the subject population or subject is        seronegative to all dengue serotypes.        8. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 7, wherein the administration of said reconstituted unit        dose and said hepatitis A vaccine is simultaneous.        9. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 8, wherein said reconstituted unit dose is administered by        subcutaneous injection and said hepatitis A vaccine is        administered by intramuscular injection, wherein said injections        are preferably administered to the arm, more preferably to the        deltoid region of the arm.        10. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 9, wherein        said reconstituted unit dose and said hepatitis A vaccine are        administered to different anatomical sites, such as to opposite        arms.        11. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 10, wherein two of said reconstituted unit doses are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        12. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 11, wherein the method comprises the administration of two        of said reconstituted unit doses and one dose of said hepatitis        A vaccine, in particular according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and said hepatitis A vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.        13. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 12, wherein the subject population or subject is of 2 to 60        years of age.        14. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 13, wherein the subject population or subject is from a        dengue endemic region.        15. The hepatitis A vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 13, wherein the subject population or subject is from a        dengue non-endemic region, preferably from a dengue non-endemic        and hepatitis-A non-endemic region.        16. A method of preventing dengue disease in a subject        population, comprising administering to the subject population a        reconstituted unit dose of any one of items 1 to 15, wherein the        method further comprises preventing hepatitis A in the subject        population by concomitant administration of a hepatitis A        vaccine, such as HAVRIX® or VAQTA®, to the subject population.        17. The method of item 16, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of a reconstituted unit dose of any one of items        17 to 19 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the GMT of dengue serotype 2 to the        GMT of dengue serotype 4.        18. The method of item 17, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        19. A method of preventing dengue disease in a subject,        comprising administering to the subject a reconstituted unit        dose of any one of items 16 to 18, wherein the method further        comprises preventing hepatitis A in the subject by concomitant        administration of a hepatitis A vaccine, such as HAVRIX® or        VAQTA®, to the subject.        20. The method of item 19, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of a reconstituted unit dose of any one of        items 17 to 19 90 days after said first administration, provide        a ratio of not more than 50, or not more than 40, or not more        than 30, or not more than 20 for the neutralizing antibody titer        of dengue serotype 2 to the neutralizing antibody titer of        dengue serotype 4.        21. The method of item 20, wherein said neutralizing antibody        titers of the subject further provide a ratio not more than 20        for the neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 1, and/or a ratio        of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        22. The method of any one of items 16 to 21, wherein the subject        population or subject is seronegative with respect to all dengue        serotypes.        23. The method of any one of items 16 to 22, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        24. The method of any one of items 16 to 23, wherein the        administration of said reconstituted unit dose and said        hepatitis A vaccine is simultaneous or sequential.        25. The method of any one of items 16 to 24, wherein said        reconstituted unit dose is administered by subcutaneous        injection and said hepatitis A vaccine is administered by        intramuscular injection, wherein said injections are preferably        administered to the arm, more preferably to the deltoid region        of the arm.        26. The method of item 25, wherein said reconstituted unit dose        and said hepatitis A vaccine are administered to different        anatomical sites, such as to opposite arms.        27. The method of any one of items 16 to 26, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        28. The method of any one of items 16 to 27, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 15 and one dose of a hepatitis A vaccine, such as        HAVRIX® or VAQTA®, in particular according to the following        schedule    -   a first simultaneous administration of the first reconstituted        unit dose and said hepatitis A vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.        29. The method of any one of items 16 to 28, wherein the subject        population or subject is of 2 to 60 years of age.        30. The method of any one of items 16 to 29, wherein the subject        population or subject is from a dengue endemic region.        31. The method of any one of items 16 to 30, wherein the subject        population or subject is from a dengue non-endemic region,        preferably from a dengue non-endemic region and a hepatitis A        non-endemic region.        32. A kit for comprising the following components:        a) a unit dose of any one of items 1 to 16,        b) a pharmaceutically acceptable diluent for reconstitution of        said unit dose, and        c) a hepatitis A vaccine.        33. The kit of item 32, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.

Sixth List of Items of the Present Invention

1. A HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9,and a reconstituted unit dose of a dengue vaccine composition for use ina method of preventing HPV-associated cancers or genital warts anddengue disease in a subject population, wherein said reconstituted unitdose comprises a tetravalent dengue virus composition including fourlive, attenuated dengue virus strains and upon reconstitution with 0.5mL of a pharmaceutically acceptable diluent comprises

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        2. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 1, wherein the        geometric mean neutralizing antibody titers (GMTs) of the        subject population when tested in at least 40, or at least 50,        or at least 60 subjects at day 180 or day 365 after at least a        first administration of said unit dose, and optionally a second        administration of said reconstituted unit dose 90 days after        said first administration, provide a ratio of not more than 50,        or not more than 40, or not more than 30, or not more than 20        for the GMT of dengue serotype 2 to the GMT of dengue serotype        4.        3. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 2, wherein said        GMTs of the subject population further provide a ratio of not        more than 20 for the GMT of dengue serotype 2 to the GMT of        dengue serotype 1, and/or a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 3.        4. A HPV vaccine, in particular a 9vHPV vaccine, such as        GARDASIL® 9, and a reconstituted unit dose of a dengue vaccine        composition for use in a method of preventing HPV-associated        cancers or genital warts and dengue disease in a subject,        wherein said reconstituted unit dose comprises a tetravalent        dengue virus composition including four live, attenuated dengue        virus strains and upon reconstitution with 0.5 mL of a        pharmaceutically acceptable diluent comprises    -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        5. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 4, wherein the        neutralizing antibody titers of the subject when tested at day        180 or day 365 after at least a first administration of said        unit dose, and optionally a second administration of said unit        dose 90 days after said first administration, provide a ratio of        not more than 50, or not more than 40, or not more than 30, or        not more than 20 for the neutralizing antibody titer of dengue        serotype 2 to the neutralizing antibody titer of dengue serotype        4.        6. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 5, wherein said        neutralizing antibody titers of the subject further provide a        ratio not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 1, and/or a ratio of not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 3.        7. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        6, wherein the subject population or subject is seronegative to        all dengue serotypes.        8. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        7, wherein the administration of said reconstituted unit dose        and said HPV vaccine is simultaneous.        9. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        8, wherein said reconstituted unit dose is administered by        subcutaneous injection and said HPV vaccine is administered by        intramuscular injection, wherein said injections are preferably        administered to the arm, more preferably to the deltoid region        of the arm.        10. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 9, wherein said        reconstituted unit dose and said HPV vaccine are administered to        different anatomical sites, such as to opposite arms.        11. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        10, wherein two of said reconstituted unit doses are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        12. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        11, wherein the method comprises the administration of two of        said reconstituted unit doses and two doses of said HPV vaccine        in particular according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said HPV vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 month        later and preferably on day 90, and    -   a third administration of the second dose of said HPV vaccine        after said second administration of the reconstituted unit dose,        such as 3 month later and preferably on day 180.        13. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        12, wherein the subject population or subject is of 9 to 25        years of age.        14. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        13, wherein the subject population or subject is from a dengue        endemic region.        15. The HPV vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        14, wherein the subject population or subject is female.        16. A method of preventing dengue disease in a subject        population, comprising administering to the subject population a        reconstituted unit dose of any one of items 1 to 15, wherein the        method further comprises preventing HPV-associated cancers or        genital warts in the subject population by concomitant        administration of a HPV vaccine, in particular a 9vHPV vaccine,        such as GARDASIL® 9, to the subject population.        17. The method of item 16, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of a reconstituted unit dose of any one of items        17 to 19 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the GMT of dengue serotype 2 to the        GMT of dengue serotype 4.        18. The method of item 17, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        19. A method of preventing dengue disease in a subject,        comprising administering to the subject a reconstituted unit        dose of any one of items 1 to 15, wherein the method further        comprises preventing HPV-associated cancers or genital warts in        the subject by concomitant administration of a HPV vaccine, in        particular a 9vHPV vaccine, such as GARDASIL® 9, to the subject.        20. The method of item 19, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of a reconstituted unit dose of any one of        items 17 to 19 90 days after said first administration, provide        a ratio of not more than 50, or not more than 40, or not more        than 30, or not more than 20 for the neutralizing antibody titer        of dengue serotype 2 to the neutralizing antibody titer of        dengue serotype 4.        21. The method of item 20, wherein said neutralizing antibody        titers of the subject further provide a ratio not more than 20        for the neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 1, and/or a ratio        of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        22. The method of any one of items 16 to 21, wherein the subject        population or subject is seronegative with respect to all dengue        serotypes.        23. The method of any one of items 16 to 22, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        24. The method of any one of items 16 to 23, wherein the        administration of said reconstituted unit dose and said HPV        vaccine is simultaneous or sequential.        25. The method of any one of items 16 to 24, wherein said        reconstituted unit dose is administered by subcutaneous        injection and said HPV vaccine is administered by intramuscular        injection, wherein said injections are preferably administered        to the arm, more preferably to the deltoid region of the arm.        26. The method of item 25, wherein said reconstituted unit dose        and said HPV vaccine are administered to different anatomical        sites, such as to opposite arms.        27. The method of any one of items 16 to 26, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        28. The method of any one of items 16 to 27, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 16 and two doses of a HPV vaccine, in particular a        9vHPV vaccine, such as GARDASIL® 9, in particular according to        the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said HPV vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 month        later and preferably on day 90, and    -   a third administration of the second dose of said HPV vaccine        after said second administration of the reconstituted unit dose,        such as 3 month later and preferably on day 180.        29. The method of any one of items 16 to 28, wherein the subject        population or subject is of 9 to 25 years of age.        30. The method of any one of items 16 to 29, wherein the subject        population or subject is from a dengue endemic region.        31. The method of any one of items 16 to 30, wherein the subject        population or subject is from a dengue non-endemic region.        32. The method of any one of items 16 to 31, wherein the subject        population or subject is female.        33. A kit for comprising the following components:        a) a unit dose of any one of items 1 to 16,        b) a pharmaceutically acceptable diluent for reconstitution of        said unit dose, and        c) a HPV vaccine.        34. The kit of item 33, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.

Seventh List of Items of the Present Invention

1. A MMR vaccine, such as M-M-R® II, and a reconstituted unit dose of adengue vaccine composition for use in a method of preventing measles,mumps and rubella and dengue disease in a subject population, whereinsaid reconstituted unit dose comprises a tetravalent dengue viruscomposition including four live, attenuated dengue virus strains andupon reconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        2. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 1, wherein the        geometric mean neutralizing antibody titers (GMTs) of the        subject population when tested in at least 40, or at least 50,        or at least 60 subjects at day 180 or day 365 after at least a        first administration of said unit dose, and optionally a second        administration of said reconstituted unit dose 90 days after        said first administration, provide a ratio of not more than 50,        or not more than 40, or not more than 30, or not more than 20        for the GMT of dengue serotype 2 to the GMT of dengue serotype        4.        3. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 2, wherein said        GMTs of the subject population further provide a ratio of not        more than 20 for the GMT of dengue serotype 2 to the GMT of        dengue serotype 1, and/or a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 3.        4. A MMR vaccine, such as M-M-R® II, and a reconstituted unit        dose of a dengue vaccine composition and a MMR vaccine, such as        M-M-R® II, for use in a method of preventing measles, mumps and        rubella and dengue disease in a subject, wherein said        reconstituted unit dose comprises a tetravalent dengue virus        composition including four live, attenuated dengue virus strains        and upon reconstitution with 0.5 mL of a pharmaceutically        acceptable diluent comprises    -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        5. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to claim 4, wherein the        neutralizing antibody titers of the subject when tested at day        180 or day 365 after at least a first administration of said        unit dose, and optionally a second administration of said unit        dose 90 days after said first administration, provide a ratio of        not more than 50, or not more than 40, or not more than 30, or        not more than 20 for the neutralizing antibody titer of dengue        serotype 2 to the neutralizing antibody titer of dengue serotype        4.        6. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to claim 5, wherein said        neutralizing antibody titers of the subject further provide a        ratio not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 1, and/or a ratio of not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 3.        7. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        6, wherein the subject population or subject is seronegative to        all dengue serotypes.        8. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        7, wherein the administration of said reconstituted unit dose        and said MMR vaccine is simultaneous or sequential.        9. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        8, wherein said reconstituted unit dose is administered by        subcutaneous injection and said MMR vaccine is administered by        subcutaneous injection, wherein said injections are preferably        administered to the arm, more preferably to the deltoid region        of the arm.        10. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 9, wherein said        reconstituted unit dose and said MMR vaccine are administered to        different anatomical sites, such as to opposite arms.        11. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of item 1 to        10, wherein two of said reconstituted unit doses are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        12. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of item 1 to        11, wherein the method comprises the administration of two of        said reconstituted unit doses and two doses of said MMR vaccine        in particular according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said MMR vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90, and    -   a third administration of the second dose of said MMR vaccine        after said second administration of the second reconstituted        unit dose, such as 3 to 6 years after said first simultaneous        administration.        13. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        12, wherein the subject population or subject is of 1 to 10        years of age.        14. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        12, wherein the subject population or subject is of 2 months to        7 years of age.        15. The MMR vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        14, wherein the subject population or subject is from a dengue        endemic region or from a dengue non-endemic region.        16. A method of preventing dengue disease in a subject        population, comprising administering to the subject population a        reconstituted unit dose of any one of items 1 to 15, wherein the        method further comprises preventing measles, mumps and rubella        in the subject population by concomitant administration of a MMR        vaccine, such as M-M-R® II, to the subject population.        17. The method of item 16, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of a reconstituted unit dose of any one of items        17 to 19 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the GMT of dengue serotype 2 to the        GMT of dengue serotype 4.        18. The method of item 17, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        19. A method of preventing dengue disease in a subject,        comprising administering to the subject a reconstituted unit        dose of any one of items 1 to 15, wherein the method further        comprises preventing measles, mumps and rubella in the subject        by concomitant administration of a MMR vaccine, such as M-M-R®        II, to the subject.        20. The method of item 19, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of a reconstituted unit dose of any one of        items 17 to 19 90 days after said first administration, provide        a ratio of not more than 50, or not more than 40, or not more        than 30, or not more than 20 for the neutralizing antibody titer        of dengue serotype 2 to the neutralizing antibody titer of        dengue serotype 4.        21. The method of item 20, wherein said neutralizing antibody        titers of the subject further provide a ratio not more than 20        for the neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 1, and/or a ratio        of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        22. The method of any one of items 16 to 21, wherein the subject        population or subject is seronegative with respect to all dengue        serotypes.        23. The method of any one of items 16 to 22, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        24. The method of any one of items 16 to 23, wherein the        administration of said reconstituted unit dose and said MMR        vaccine is simultaneous or sequential.        25. The method of any one of items 16 to 23, wherein said        reconstituted unit dose is administered by subcutaneous        injection and said MMR vaccine is administered by subcutaneous        injection, wherein said injections are preferably administered        to the arm, more preferably to the deltoid region of the arm.        26. The method of item 25, wherein said reconstituted unit dose        and said MMR vaccine are administered to different anatomical        sites, such as to opposite arms.        27. The method of any one of items 16 to 26, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        28. The method of any one of items 16 to 27, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 15 and two doses of a MMR vaccine, such as M-M-R® II,        in particular according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said MMR vaccine on day 0,    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90, and    -   a third administration of the second dose of said MMR vaccine        after said second administration of the second reconstituted        unit dose, such as 3 to 6 years after said first simultaneous        administration.        29. The method of any one of items 16 to 28, wherein the subject        population or subject is of 1 to 10 years of age, preferably of        2 months to 7 years of age.        30. The method of any one of items 16 to 29, wherein the subject        population or subject is from a dengue endemic region.        31. The method of any one of items 16 to 30, wherein the subject        population or subject is from a dengue non-endemic region.        32. A kit for comprising the following components:        a) a unit dose of any one of items 1 to 15,        b) a pharmaceutically acceptable diluent for reconstitution of        said unit dose, and        c) a MMR vaccine.        33. The kit of item 32, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.

Eighth List of Items of the Present Invention

1. A tetanus, diphtheria, and pertussis (Tdap) vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, and a reconstitutedunit dose of a dengue vaccine composition for use in a method ofpreventing tetanus, diphtheria, and pertussis and dengue disease in asubject population, wherein said reconstituted unit dose comprises atetravalent dengue virus composition including four live, attenuateddengue virus strains and upon reconstitution with 0.5 mL of apharmaceutically acceptable diluent comprises(i) a chimeric dengue serotype 2/1 strain in a concentration of at least3.3 log 10 pfu/0.5 mL,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 log10 pfu/0.5 mL,(iii) a chimeric dengue serotype 2/3 strain in a concentration of atleast 4.0 log 10 pfu/0.5 mL, and(iv) a chimeric dengue serotype 2/4 strain in a concentration of atleast 4.5 log 10 pfu/0.5 mL.2. The Tdap vaccine and the reconstituted unit dose of a dengue vaccinecomposition for use according to item 1, wherein the geometric meanneutralizing antibody titers (GMTs) of the subject population whentested in at least 40, or at least 50, or at least 60 subjects at day180 or day 365 after at least a first administration of said unit dose,and optionally a second administration of said reconstituted unit dose90 days after said first administration, provide a ratio of not morethan 50, or not more than 40, or not more than 30, or not more than 20for the GMT of dengue serotype 2 to the GMT of dengue serotype 4.3. The Tdap vaccine and the reconstituted unit dose of a dengue vaccinecomposition for use according to item 2, wherein said GMTs of thesubject population further provide a ratio of not more than 20 for theGMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or a ratioof not more than 20 for the GMT of dengue serotype 2 to the GMT ofdengue serotype 3.4. A tetanus, diphtheria, and pertussis (Tdap) vaccine, in particular acombined tetanus toxoid, reduced diphtheria toxoid and acellularpertussis (adsorbed) vaccine, such as BOOSTRIX®, and a reconstitutedunit dose of a dengue vaccine composition for use in a method ofpreventing tetanus, diphtheria, and pertussis and dengue disease in asubject, wherein said reconstituted unit dose comprises a tetravalentdengue virus composition including four live, attenuated dengue virusstrains and upon reconstitution with 0.5 mL of a pharmaceuticallyacceptable diluent comprises

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and    -   (iv) a chimeric dengue serotype 2/4 strain in a concentration of        at least 4.5 log 10 pfu/0.5 mL.        5. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 4, wherein the        neutralizing antibody titers of the subject when tested at day        180 or day 365 after at least a first administration of said        unit dose, and optionally a second administration of said unit        dose 90 days after said first administration, provide a ratio of        not more than 50, or not more than 40, or not more than 30, or        not more than 20 for the neutralizing antibody titer of dengue        serotype 2 to the neutralizing antibody titer of dengue serotype        4.        6. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 5, wherein said        neutralizing antibody titers of the subject further provide a        ratio not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 1, and/or a ratio of not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 3.        7. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        6, wherein the subject population or subject is seronegative to        all dengue serotypes.        8. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        7, wherein the administration of said reconstituted unit dose        and said Tdap vaccine is simultaneous.        9. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        8, wherein said reconstituted unit dose is administered by        subcutaneous injection and said Tdap vaccine is administered by        intramuscular injection, wherein said injections are preferably        administered to the arm, more preferably to the deltoid region        of the arm.        10. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to item 9, wherein said        reconstituted unit dose and said Tdap vaccine are administered        to different anatomical sites, such as to opposite arms.        11. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        10, wherein two of said reconstituted unit doses are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        12. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        11, wherein the method comprises the administration of two of        said reconstituted unit doses and one dose of said Tdap vaccine        in particular according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and said Tdap vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.        13. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        12, wherein the subject population or subject is of 10 to 18        years of age.        14. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        13, wherein the subject population or subject is from a dengue        endemic region.        15. The Tdap vaccine and the reconstituted unit dose of a dengue        vaccine composition for use according to any one of items 1 to        13, wherein the subject population or subject is from a dengue        non-endemic region.        16. A method of preventing dengue disease in a subject        population, comprising administering to the subject population a        reconstituted unit dose of any one of items 1 to 15, wherein the        method further comprises preventing tetanus, diphtheria, and        pertussis in the subject population by concomitant        administration of a tetanus, diphtheria, and pertussis (Tdap)        vaccine, in particular a combined tetanus toxoid, reduced        diphtheria toxoid and acellular pertussis (adsorbed) vaccine,        such as BOOSTRIX®, to the subject population.        17. The method of item 16, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of a reconstituted unit dose of any one of items        17 to 19 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the GMT of dengue serotype 2 to the        GMT of dengue serotype 4.        18. The method of item 17, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        19. A method of preventing dengue disease in a subject,        comprising administering to the subject a reconstituted unit        dose of any one of items 1 to 15, wherein the method further        comprises preventing tetanus, diphtheria, and pertussis in the        subject by concomitant administration of a tetanus, diphtheria,        and pertussis (Tdap) vaccine, in particular a combined tetanus        toxoid, reduced diphtheria toxoid and acellular pertussis        (adsorbed) vaccine, such as BOOSTRIX®, to the subject.        20. The method of item 19, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of a reconstituted unit dose of any one of        items 1 to 15 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 4.        21. The method of item 20, wherein said neutralizing antibody        titers of the subject further provide a ratio not more than 20        for the neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 1, and/or a ratio        of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        22. The method of any one of items 16 to 21, wherein the subject        population or subject is seronegative with respect to all dengue        serotypes.        23. The method of any one of items 16 to 22, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        24. The method of any one of items 16 to 23, wherein the        administration of said reconstituted unit dose and said Tdap        vaccine is simultaneous or sequential.        25. The method of any one of items 16 to 24, wherein said        reconstituted unit dose is administered by subcutaneous        injection and said Tdap vaccine is administered by intramuscular        injection, wherein said injections are preferably administered        to the arm, more preferably to the deltoid region of the arm.        26. The method of item 25, wherein said reconstituted unit dose        and said Tdap vaccine are administered to different anatomical        sites, such as to opposite arms.        27. The method of any one of items 16 to 26, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        28. The method of any one of items 16 to 27, wherein the method        comprises the administration of two reconstituted unit doses of        items 1 to 15 and one dose of a Tdap vaccine, in particular a        combined tetanus toxoid, reduced diphtheria toxoid and acellular        pertussis (adsorbed) vaccine, such as BOOSTRIX®, in particular        according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and said Tdap vaccine on day 0, and    -   a second administration of the second reconstituted unit dose        after said first simultaneous administration, such as 3 months        later and preferably on day 90.        29. The method of any one of items 16 to 28, wherein the subject        population or subject is of 10 to 18 years of age.        30. The method of any one of items 16 to 29, wherein the subject        population or subject is from a dengue endemic region.        31. The method of any one of items 16 to 29, wherein the subject        population or subject is from a dengue non-endemic region.        32. A kit for comprising the following components:        a) a unit dose of any one of items 1 to 15,        b) a pharmaceutically acceptable diluent for reconstitution of        said unit dose, and        c) a Tdap vaccine.        33. The kit of item 32, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.

Ninth List of Items of the Present Invention

1. A DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hibvaccine, such as Pentacel®, and a reconstituted unit dose of a denguevaccine composition for use in a method of preventing diphtheria,tetanus, pertussis, poliomyelitis and diseases caused by Haemophilusinfluenzae type b and dengue disease in a subject population, whereinsaid reconstituted unit dose comprises a tetravalent dengue viruscomposition including four live, attenuated dengue virus strains andupon reconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises(i) a chimeric dengue serotype 2/1 strain in a concentration of at least3.3 log 10 pfu/0.5 mL,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 log10 pfu/0.5 mL,(iii) a chimeric dengue serotype 2/3 strain in a concentration of atleast 4.0 log 10 pfu/0.5 mL, and(iv) a chimeric dengue serotype 2/4 strain in a concentration of atleast 4.5 log 10 pfu/0.5 mL.2. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a denguevaccine composition for use according to item 1, wherein the geometricmean neutralizing antibody titers (GMTs) of the subject population whentested in at least 40, or at least 50, or at least 60 subjects at day180 or day 365 after at least a first administration of said unit dose,and optionally a second administration of said reconstituted unit dose90 days after said first administration, provide a ratio of not morethan 50, or not more than 40, or not more than 30, or not more than 20for the GMT of dengue serotype 2 to the GMT of dengue serotype 4.3. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a denguevaccine composition for use according to item 2, wherein said GMTs ofthe subject population further provide a ratio of not more than 20 forthe GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or aratio of not more than 20 for the GMT of dengue serotype 2 to the GMT ofdengue serotype 3.4. A DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hibvaccine, such as Pentacel®, and a reconstituted unit dose of a denguevaccine composition for use in a method of preventing diphtheria,tetanus, pertussis, poliomyelitis and diseases caused by Haemophilusinfluenzae type b and dengue disease in a subject, wherein saidreconstituted unit dose comprises a tetravalent dengue virus compositionincluding four live, attenuated dengue virus strains and uponreconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises

-   -   (i) a chimeric dengue serotype 2/1 strain in a concentration of        at least 3.3 log 10 pfu/0.5 mL,    -   (ii) a dengue serotype 2 strain in a concentration of at least        2.7 log 10 pfu/0.5 mL,    -   (iii) a chimeric dengue serotype 2/3 strain in a concentration        of at least 4.0 log 10 pfu/0.5 mL, and (iv) a chimeric dengue        serotype 2/4 strain in a concentration of at least 4.5 log 10        pfu/0.5 mL.        5. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 4, wherein        the neutralizing antibody titers of the subject when tested at        day 180 or day 365 after at least a first administration of said        unit dose, and optionally a second administration of said unit        dose 90 days after said first administration, provide a ratio of        not more than 50, or not more than 40, or not more than 30, or        not more than 20 for the neutralizing antibody titer of dengue        serotype 2 to the neutralizing antibody titer of dengue serotype        4.        6. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to item 5, wherein        said neutralizing antibody titers of the subject further provide        a ratio not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 1, and/or a ratio of not more than 20 for the        neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 3.        7. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 6, wherein the subject population or subject is        seronegative to all dengue serotypes.        8. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 7, wherein the administration of said reconstituted unit        dose and said DTaP/IPV/Hib vaccine is simultaneous or        sequential.        9. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of a        dengue vaccine composition for use according to any one of items        1 to 8, wherein said reconstituted unit dose is administered by        subcutaneous injection and said DTaP/IPV/Hib vaccine is        administered by intramuscular injection, wherein said injections        are preferably administered to the arm, more preferably to the        deltoid region of the arm.        10. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to item 9,        wherein said reconstituted unit dose and said DTaP/IPV/Hib        vaccine are administered to different anatomical sites, such as        to arm and thigh.        11. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to item 9 or 10,        wherein said subcutaneous injection of said unit dose is        administered to the arm, preferably to the deltoid region of the        arm, and said intramuscular injection of said DTaP/IPV/Hib        vaccine is administered to the thigh, preferably to the        anterolateral aspect of the thigh.        12. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 11, wherein two of said reconstituted unit doses are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        13. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 12, wherein the method comprises the administration        of two of said reconstituted unit doses and four doses of said        DTaP/IPV/Hib vaccine in particular according to the following        schedule    -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said DTaP/IPV/Hib vaccine on day        0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first simultaneous administration, such as 2        months later and preferably on day 60,    -   a third administration of the second reconstituted unit dose        after said second administration, such as 3 months after the        first simultaneous administration and preferably on day 90,    -   a fourth administration of the third dose of said DTaP/IPV/Hib        vaccine after said third administration, such as 4 months after        the first simultaneous administration and preferably on day 120,        and    -   a fifth administration of the fourth dose of said DTaP/IPV/Hib        vaccine after said fourth administration, such as 9 to 12 months        later and preferably on day 390.        14. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 12, wherein the method comprises the administration        of two of said reconstituted unit doses and four doses of said        DTaP/IPV/Hib vaccine in particular according to the following        schedule    -   a first administration of the first dose of said DTaP/IPV/Hib        vaccine on day 0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first administration, such as 2 months later        and preferably on day 60,    -   a third administration of the third dose of said DTaP/IPV/Hib        vaccine after said second administration, such as 4 months after        the first administration and preferably on day 120,    -   a fourth simultaneous administration of the first reconstituted        unit dose and the fourth dose of said DTaP/IPV/Hib vaccine after        said third administration, such as 9 to 12 months later and        preferably on day 390, and    -   a fifth administration of the second reconstituted unit dose        after said fourth simultaneous administration, such as 3 months        later and preferably on day 480.        15. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to item 13 or 14,        wherein a fifth dose of a DTaP/IPV/Hib vaccine, in particular a        combined DTaP/IPV/Hib vaccine, such as Quadracel®, is        administered after the administration of the fourth dose of said        DTaP/IPV/Hib vaccine, such as 30 to 57 months after the        administration of the fourth dose of a DTaP/IPV/Hib vaccine,        such as Pentacel®.        16. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 15, wherein the subject population or subject is of 2        months to 4 years of age.        17. The DTaP/IPV/Hib vaccine and the reconstituted unit dose of        a dengue vaccine composition for use according to any one of        items 1 to 16, wherein the subject population or subject is from        a dengue endemic region or from a dengue non-endemic region.        18. A method of preventing dengue disease in a subject        population, comprising administering to the subject population a        reconstituted unit dose of any one of items 1 to 15, wherein the        method further comprises preventing diphtheria, tetanus,        pertussis, poliomyelitis and diseases caused by Haemophilus        influenzae type b in the subject population by concomitant        administration of a DTaP/IPV/Hib vaccine, in particular a        combined DTaP/IPV/Hib vaccine, such as Pentacel®, to the subject        population.        19. The method of item 18, wherein the geometric mean        neutralizing antibody titers (GMTs) of the subject population        when tested in at least 40, or at least 50, or at least 60        subjects at day 180 or day 365 after at least a first        administration of said unit dose, and optionally a second        administration of a reconstituted unit dose of any one of items        17 to 19 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the GMT of dengue serotype 2 to the        GMT of dengue serotype 4.        20. The method of item 19, wherein said GMTs of the subject        population further provide a ratio of not more than 20 for the        GMT of dengue serotype 2 to the GMT of dengue serotype 1, and/or        a ratio of not more than 20 for the GMT of dengue serotype 2 to        the GMT of dengue serotype 3.        21. A method of preventing dengue disease in a subject,        comprising administering to the subject a reconstituted unit        dose of any one of items 1 to 15, wherein the method further        comprises preventing diphtheria, tetanus, pertussis,        poliomyelitis and diseases caused by Haemophilus influenzae type        b in the subject by concomitant administration of a DTaP/IPV/Hib        vaccine, in particular a combined DTaP/IPV/Hib vaccine, such as        Pentacel®, to the subject.        22. The method of item 21, wherein the neutralizing antibody        titers of the subject when tested at day 180 or day 365 after at        least a first administration of said unit dose, and optionally a        second administration of a reconstituted unit dose of any one of        items 1 to 15 90 days after said first administration, provide a        ratio of not more than 50, or not more than 40, or not more than        30, or not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 4.        23. The method of item 22, wherein said neutralizing antibody        titers of the subject further provide a ratio not more than 20        for the neutralizing antibody titer of dengue serotype 2 to the        neutralizing antibody titer of dengue serotype 1, and/or a ratio        of not more than 20 for the neutralizing antibody titer of        dengue serotype 2 to the neutralizing antibody titer of dengue        serotype 3.        24. The method of any one of items 18 to 23, wherein the subject        population or subject is seronegative with respect to all dengue        serotypes.        25. The method of any one of items 18 to 24, wherein the subject        population or subject is seropositive with respect to at least        one dengue serotype.        26. The method of any one of items 18 to 25, wherein the        administration of said reconstituted unit dose and said        DTaP/IPV/Hib vaccine is simultaneous or sequential.        27. The method of any one of items 18 to 26, wherein said        reconstituted unit dose is administered by subcutaneous        injection and said DTaP/IPV/Hib vaccine is administered by        intramuscular injection.        28. The method of item 27, wherein said reconstituted unit dose        and said DTaP/IPV/Hib vaccine are administered to different        anatomical sites, such as to arm and thigh.        29. The method of item 27 or 28, wherein said subcutaneous        injection is administered to the arm, preferably to the deltoid        region of the arm, and said intramuscular injection is        administered to the thigh, preferably to the anterolateral        aspect of the thigh.        30. The method of any one of items 18 to 29, wherein two        reconstituted unit doses of any one of items 17 to 19 are        administered within 12 months or more, or within six months, or        within three months, such as at day 0 and day 90.        31. The method of any one of items 18 to 30, wherein the method        comprises the administration of two reconstituted unit doses of        items 17 to 19 and four doses of a DTaP/IPV/Hib vaccine, in        particular a combined DTaP/IPV/Hib vaccine, such as Pentacel®,        in particular according to the following schedule    -   a first simultaneous administration of the first reconstituted        unit dose and the first dose of said DTaP/IPV/Hib vaccine on day        0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first simultaneous administration, such as 2        months later and preferably on day 60,    -   a third administration of the second reconstituted unit dose        after said second administration, such as 3 months after the        first simultaneous administration and preferably on day 90,    -   a fourth administration of the third dose of said DTaP/IPV/Hib        vaccine after said third administration, such as 4 months after        the first simultaneous administration and preferably on day 120,        and    -   a fifth administration of the fourth dose of said DTaP/IPV/Hib        vaccine after said fourth administration, such as 9 to 12 months        later and preferably on day 390.        32. The method of any one of items 18 to 31, wherein the method        comprises the administration of two reconstituted unit doses of        items 17 to 19 and four doses of a DTaP/IPV/Hib vaccine, in        particular a combined DTaP/IPV/Hib vaccine, such as Pentacel®,        in particular according to the following schedule    -   a first administration of the first dose of said DTaP/IPV/Hib        vaccine on day 0,    -   a second administration of the second dose of said DTaP/IPV/Hib        vaccine after said first administration, such as 2 months later        and preferably on day 60,    -   a third administration of the third dose of said DTaP/IPV/Hib        vaccine after said second administration, such as 4 months after        the first administration and preferably on day 120,    -   a fourth simultaneous administration of the first reconstituted        unit dose and the fourth dose of said DTaP/IPV/Hib vaccine after        said third administration, such as 9 to 12 months later and        preferably on day 390, and    -   a fifth administration of the second reconstituted unit dose        after said fourth simultaneous administration, such as 3 months        later and preferably on day 480.        33. The method of item 18 to 32, wherein a fifth dose of a        DTaP/IPV/Hib vaccine, in particular a combined DTaP/IPV/Hib        vaccine, such as Quadracel®, is administered after the        administration of the fourth dose of said DTaP/IPV/Hib vaccine,        such as 30 to 57 months after the administration of the fourth        dose of a DTaP/IPV/Hib vaccine, in particular after the fourth        dose of a combined DTaP/IPV/Hib vaccine, such as Pentacel®.        34. The method of any one of items 18 to 33, wherein the subject        population or subject is of 2 months to 4 years of age.        35. The method of any one of items 18 to 34, wherein the subject        population or subject is from a dengue endemic region.        36. The method of any one of items 18 to 35, wherein the subject        population or subject is from a dengue non-endemic region.        37. A kit for comprising the following components: a) a unit        dose of any one of items 1 to 15, b) a pharmaceutically        acceptable diluent for reconstitution of said unit dose, and c)        a DTaP/IPV/Hib vaccine.        38. The kit of 37, wherein the pharmaceutically acceptable        diluent for reconstitution is 37 mM sodium chloride.

Tenth List of Items of the Present Invention

1. A reconstituted unit dose of a dengue vaccine composition for use ina method of preventing dengue disease in an elderly subject, whereinsaid reconstituted unit dose comprises a tetravalent dengue viruscomposition including four live, attenuated dengue virus strains andupon reconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises(i) a chimeric dengue serotype 2/1 strain in a concentration of at least3.3 log 10 pfu/0.5 mL,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 log10 pfu/0.5 mL,(iii) a chimeric dengue serotype 2/3 strain in a concentration of atleast 4.0 log 10 pfu/0.5 mL, and(iv) a chimeric dengue serotype 2/4 strain in a concentration of atleast 4.5 log 10 pfu/0.5 mL.2. The reconstituted unit dose of a dengue vaccine composition for useaccording to item 1, wherein dengue hemorrhagic fever (DHF) and dengueshock syndrome (DSS) are prevented in the elderly subject.3. The reconstituted unit dose of a dengue vaccine composition for useaccording to item 1 or 2, wherein the reconstituted unit dose isadministered by subcutaneous injection, preferably to the deltoid regionof the arm.4. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 3, wherein two of said reconstitutedunit doses are administered.5. The reconstituted unit dose of a dengue vaccine composition for useaccording to item 4, wherein said two unit doses are administered within12 months, preferably within 3 months, such as at about day 0 and aboutday 90.6. The reconstituted unit dose of a dengue vaccine composition for useaccording to item 4 or 5, wherein a third unit dose is administeredafter administration of said second unit dose.7. The reconstituted unit dose of a dengue vaccine composition for useaccording to item 6, wherein said third unit dose is administeredbetween 6 to 12 months after the first administration, such as 12 monthafter the first administration.8. The reconstituted unit dose of a dengue vaccine composition for useaccording to claim 6, wherein said third unit dose is administered laterthan 12 months after the first administration, such as 12 months afterthe second administration.9. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 8, wherein the elderly subject isfrom a dengue endemic region.10. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 8, wherein the elderly subject isfrom a dengue non-endemic region.11. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 10, wherein the elderly subject isseronegative with respect to all dengue serotypes.12. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 10, wherein the elderly subject isseropositive with respect to at least one dengue serotype.13. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 12, wherein the elderly subject hasat least one chronic condition or disease.14. The reconstituted unit dose of a dengue vaccine composition for useaccording to item 13, wherein the at least one chronic condition ordisease is selected from diabetes, hypertension, allergies, previousstrokes, ischemic heart disease, chronic renal impairment and chronicobstructive pulmonary disease.15. The reconstituted unit dose of a dengue vaccine composition for useaccording to any one of items 1 to 14, wherein the elderly subject hasan impaired immune system.16. A method of preventing dengue disease in an elderly subjectcomprising administering to the elderly subject a reconstituted unitdose of any one of items 1 to 15.17. A method of preventing dengue hemorrhagic fever (DHF) or dengueshock syndrome (DSS) in an elderly subject comprising administering tothe elderly subject a reconstituted unit dose of any one of items 1 to15.18. The method of items 16 or 17, wherein the reconstituted unit dose isadministered by subcutaneous injection, preferably to the deltoid regionof the arm.19. The method of any one of items 16 to 18, wherein two reconstitutedunit doses of any one of items 17 to 19 are administered within 12months or more.20. The method of any one of items 16 to 19, wherein two reconstitutedunit doses of any one of items 17 to 19 are administered within sixmonths, preferably within three months.21. The method of item 20, wherein the two reconstituted unit doses areadministered at day 0 and day 90.22. The method of any one of items 16 to 21, wherein a third unit doseis administered after administration of the second unit dose, preferablywithin 12 months after administration of the first unit dose.23. The method of any one of items 16 to 21, wherein a third unit doseis administered after administration of the second unit dose, preferablywithin 12 months after administration of the second unit dose.24. The method of any one of items 16 to 24, wherein the elderly subjectis seronegative with respect to all dengue serotypes.25. The method of any one of items 16 to 24, wherein the elderly subjectis seropositive with respect to at least one dengue serotypes.26. The method of any one of items 16 to 25, wherein the elderly subjectis of more than 60 years of age.27. The method of any one of items 16 to 26, wherein the elderly subjectis from a dengue endemic region.28. The method of any one of items 16 to 27, wherein the elderly subjectis from a dengue non-endemic region.29. The method of any one of items 16 to 28, wherein the elderly subjecthas at least one chronic condition or disease.

30. The method of item 29, wherein the at least one chronic condition ordisease is selected from diabetes, hypertension, allergies, previousstrokes, ischemic heart disease, chronic renal impairment and chronicobstructive pulmonary disease.

31. The method of any one of items 16 to 30, wherein the elderly subjecthas an impaired immune system.32. A method for stimulating an immune response to all four serotypes ofdengue virus in an elderly subject, comprising administering to theelderly subject a reconstituted unit dose of items 1 to 15.33. The method of item 32, wherein the immune response to all fourserotypes of dengue virus is balanced.34. The method of items 32 or 33, wherein the reconstituted unit dose isadministered by subcutaneous injection, preferably to the deltoid regionof the arm.35. The method of any one of items 32 to 34, wherein two unit doses ofany one of items 17 to 19 are administered within 12 months or more.36. The method of any one of items 32 to 35, wherein two reconstitutedunit doses of any one of items 17 to 19 are administered within sixmonths, preferably within three months.37. The method of item 36, wherein the two reconstituted unit doses areadministered at day 0 and day 90.38. The method of any one of items 32 to 37, wherein a third unit doseis administered after the administration of the second unit dose,preferably within 12 month of administration of the first unit dose.39. The method of any one of items 32 to 38, wherein a third unit doseis administered after the administration of the second unit dose,preferably within 12 month of administration of the second unit dose.40. The method of any one of items 32 to 39, wherein the elderly subjectis from a dengue endemic region.41. The method of any one of items 32 to 40, wherein the elderly subjectis from a dengue non-endemic region.42. The method of any one of items 32 to 41, wherein the elderly subjectis seronegative with respect to all dengue serotypes.43. The method of any one of items 32 to 42, wherein the elderly subjectis seropositive with respect to at least one dengue serotype.44. The method of any one of items 32 to 43, wherein the elderly subjecthas at least one chronic condition or disease.45. The method of item 44, wherein the at least one chronic condition ordisease is selected from diabetes, hypertension, allergies, previousstrokes, ischemic heart disease, chronic renal impairment and chronicobstructive pulmonary disease.46. The method of any one of items 32 to 45, wherein the elderly subjecthas an impaired immune system.47. The method of any one of items 17 to 47, wherein the reconstitutedunit dose is obtained from the kit according to item 50 or 51.48. The reconstituted unit dose of any one of items 1 to 16 for use in amethod of items 17 to 54.49. Use of a reconstituted unit dose of any one of items 1 to 16 for themanufacture of a medicament for a method according to items 17 to 48.50. A kit for preparing a reconstituted unit dose comprising thefollowing components:

a) a unit dose of any one of items 1 to 16, and

b) a pharmaceutically acceptable diluent for reconstitution.

51. The kit of item 50, wherein the pharmaceutically acceptable diluentfor reconstitution is 37 mM sodium chloride.

1. A unit dose of a dengue vaccine composition comprising: a tetravalentvirus composition including four live attenuated dengue virus serotypeswherein the unit dose is lyophilized and upon reconstitution with 0.5 mLof a pharmaceutically acceptable diluent comprises: (i) a dengueserotype 1, such as a chimeric dengue serotype 2/1 strain, in aconcentration of at least 3.3 log 10 pfu/0.5 ml, (ii) a dengue serotype2, such as a dengue serotype 2 strain, in a concentration of at least2.7 log 10 pfu/0.5 ml, (iii) a dengue serotype 3, such as a chimericdengue serotype 2/3 strain, in a concentration of at least 4.0 log 10pfu/0.5 ml, and (IV) a dengue serotype 4, such as a chimeric dengueserotype 2/4 strain, in a concentration of at least 4.5 log 10 pfu/0.5ml.
 2. The unit dose according to claim 1, wherein the dengue serotype 2strain is derived from the wild type virus strain DEN-2 16681 anddiffers in at least three nucleotides from the wild type as follows: a)5′-noncoding region (NCR)-57 (nt-57 C-to-T) b) NS1-53 Gly-to-Asp(nt-2579 G-to-A) c) NS3-250 Glu-to-Val (nt-5270 A-to-T); and wherein thethree chimeric dengue strains are derived from the serotype 2 strain byreplacing the structural proteins prM and E from serotype 2 strain withthe corresponding structural proteins from the other dengue serotypes,resulting in the following chimeric dengue strains: a DENV-2/1 chimera,a DENV-2/3 chimera and a DENV-2/4 chimera.
 3. The unit dose according toclaim 1, wherein upon reconstitution with a pharmaceutically acceptablediluent, i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration of pfu/0.5 ml theconcentration of (ii) in pfu/0.5 mL is less than 10%, and theconcentration of (iv) in pfu/0.5 mL is at least 50%, and theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 8%, or at least 10%, or at least 12%,or at least 14%, or at least 16%, or at least 18%.
 4. The unit doseaccording to claim 3, wherein the concentration of (iii) in pfu/0.5 mLis at least 10%.
 5. The unit dose according to claim 1, wherein uponreconstitution with a pharmaceutically acceptable diluent, i), (ii),(iii), and (iv) provide a total concentration of pfu/0.5 mL and based onsaid total concentration the concentration of (ii) in pfu/0.5 mL is lessthan 2%, the concentration of (iv) in pfu/0.5 mL is at least 50%, theconcentration of (i) in pfu/0.5 mL is at least 1%, and the concentrationof (iii) in pfu/0.5 mL is at least 6%. 6.-25. (canceled)
 26. A method ofinoculating a subject against virologically confirmable dengue diseasewith a tetravalent dengue virus composition including four liveattenuated dengue virus strains representing serotype 1, serotype 2,serotype 3 and serotype
 4. 27. The method according to claim 26, whereinthe tetravalent dengue virus composition includes a chimeric dengueserotype 2/1 strain and a dengue serotype 2 strain and a chimeric dengueserotype 2/3 strain and a chimeric dengue serotype 2/4 strain.
 28. Themethod according to claim 27, wherein the dengue serotype 2 strain isderived from the wild type virus strain DEN-2 16681 and differs in atleast three nucleotides from the wild type as follows: a) 5′-noncodingregion (NCR)-57 (nt-57 C-to-T) b) NS1-53 Gly-to-Asp (nt-2579 G-to-A) c)NS3-250 Glu-to-Val (nt-5270 A-to-T); and wherein the three chimericdengue strains are derived from the serotype 2 strain by replacing thestructural proteins prM and E from serotype 2 strain with thecorresponding structural proteins from the other dengue serotypes,resulting in the following chimeric dengue strains: a DENV-2/1 chimera,a DENV-2/3 chimera and a DENV-2/4 chimera.
 29. The method according toclaim 28, wherein the tetravalent dengue virus composition is in theform of a unit dose comprising: (i) a dengue serotype 1 in aconcentration of at least 3.3 log 10 pfu/0.5 ml, (ii) a dengue serotype2, in a concentration of at least 2.7 log 10 pfu/0.5 ml, (iii) a dengueserotype 3, in a concentration of at least 4.0 log 10 pfu/0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 log 10pfu/0.5 ml.
 30. The method according to claim 28, wherein tetravalentdengue virus composition is in the form of a lyophilized unit dose whichupon reconstitution with 0.5 mL of a pharmaceutically acceptable diluentcomprises: (i) a dengue serotype 1 in a concentration of at least 3.3log 10 pfu/0.5 ml, (ii) a dengue serotype 2, in a concentration of atleast 2.7 log 10 pfu/0.5 ml, (iii) a dengue serotype 3, in aconcentration of at least 4.0 log 10 pfu/0.5 ml, and (iv) a dengueserotype 4, in a concentration of at least 4.5 log 10 pfu/0.5 ml. 31.The method according to claim 28, wherein the method comprises a primaryvaccination with only two administrations of the unit dose comprisingthe steps of: administering a first unit dose of the tetravalent denguevirus composition to the subject, and administering a second unit doseof the tetravalent dengue virus composition to the subject within 3months of administration of the first unit dose.
 32. The methodaccording to claim 31, wherein the method does not include a step ofdetermination whether there was a previous dengue infection in thesubject before administration of the unit dose or wherein the serostatusof the subject is unknown before administration of the unit dose. 33.The method according to claim 31, wherein the method does not include astep of determination whether there was a previous dengue infection inthe subject at any time before, during or after the steps ofadministration of the unit dose or wherein the serostatus of the subjectis unknown before, during or after administration of the unit dose. 34.The method according to claim 28, wherein the method comprises a primaryvaccination consisting of the steps of: (A) selecting a subject foradministration of the unit doses of the tetravalent dengue viruscomposition in need for protection against dengue infection withoutdetermination whether there was a previous dengue infection, and (B)administering a first unit dose of the tetravalent dengue viruscomposition to the subject, and (C) administering a second unit dose ofthe tetravalent dengue virus composition to the subject within 3 monthsof administration of the first unit dose.
 35. The method according toclaim 28, wherein the subject is from a region wherein theseroprevalence rate is unknown and/or wherein the seroprevalence isbelow 80%, or below 70%, or below 60%.
 36. The method according to claim28, wherein the subject is exposed to a dengue outbreak.
 37. The methodaccording to claim 36, wherein the outbreak is due to a dengue serotype2, and/or due to a dengue serotype
 1. 38. The method according to claim28, wherein the virologically confirmable dengue disease is due to adengue serotype 2, and/or due to a dengue serotype
 1. 39. The methodaccording to claim 28, wherein the subject is under 9 years of age, 4 to5 years of age, 6 to 11 years of age or 12 to 16 years, or 6 to 16 yearsof age, or 4 to 16 years of age, or 2 to 17 years of age, or 9 years ofage, or over 9 years of age, or 9 to 19 years of age, or 18 to 60 yearsof age, or 18 to 45 years of age, or 46 to 60 years of age, or over 60years of age.
 40. A method of inoculating a subject againstvirologically confirmable dengue disease with a tetravalent dengue viruscomposition including four live attenuated dengue virus strainsrepresenting serotype 1, serotype 2, serotype 3 and serotype 4, thedengue serotype 2 strain being derived from the wild type virus strainDEN-2 16681 and differing in at least three nucleotides from the wildtype as follows: a) 5′-noncoding region (NCR)-57 (nt-57 C-to-T) b)NS1-53 Gly-to-Asp (nt-2579 G-to-A) c) NS3-250 Glu-to-Val (nt-5270A-to-T); and the three chimeric dengue strains being derived from theserotype 2 strain by replacing the structural proteins prM and E fromserotype 2 strain with the corresponding structural proteins from theother dengue serotypes, resulting in the following chimeric denguestrains: a DENV-2/1 chimera, a DENV-2/3 chimera and a DENV-2/4 chimera,the method comprises a primary vaccination consisting of the steps of:(A) selecting a subject for administration of the unit doses of thetetravalent dengue virus composition in need for protection againstdengue infection without determination whether there was a previousdengue infection, and (B) administering a first unit dose of thetetravalent dengue virus composition to the subject, and (C)administering a second unit dose of the tetravalent dengue viruscomposition to the subject within 3 months of administration of thefirst unit dose.
 41. The method according to claim 40, wherein thesubject is from a region wherein the seroprevalence rate is unknownand/or wherein the seroprevalence is below 80%, or below 70%, or below60%.
 42. The method according to claim 40, wherein the subject is under9 years of age, 4 to 5 years of age, 6 to 11 years of age or 12 to 16years, or 6 to 16 years of age, or 4 to 16 years of age, or 2 to 17years of age, or 9 years of age, or over 9 years of age, or 9 to 19years of age, or 18 to 60 years of age, 18 to 45 years of age, or 46 to60 years of age, or over 60 years of age.
 43. The method according toclaim 40, wherein upon reconstitution with a pharmaceutically acceptablediluent (i), (ii), (iii), and (iv) provide a total concentration ofpfu/0.5 mL and based on said total concentration the concentration of(ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) inpfu/0.5 mL is at least 50%, and the concentration of (i) in pfu/0.5 mLis at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%,or at least 18%, and wherein preferably the subject is 2 to 17 years ofage or 4 to 16 years of age.
 44. The method according to claim 40,wherein upon reconstitution with a pharmaceutically acceptable diluent(i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mLand based on said total concentration the concentration of (ii) inpfu/0.5 mL is less than 2%, the concentration of (iv) in pfu/0.5 mL isat least 50%, the concentration of (i) in pfu/0.5 mL is at least 1%, andthe concentration of (iii) in pfu/0.5 mL is at least 6%, whereinpreferably the subject is 18 to 60 years of age.
 45. The method of claim40 which is safe.
 46. The method of claim 45, providing a combinedvaccine efficacy against virologically-confirmed dengue withhospitalization against all four serotypes with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 65%, when measuredagainst placebo in a subject population of at least 5,000 healthy 4 to16 year old subjects irrespective of serostatus at baseline from firstadministration of the administration schedule until 12 to 18 monthsafter the second administration of the administration schedule.
 47. Themethod of claim 40 which is effective.
 48. The method of claim 47,providing a combined vaccine efficacy against all four serotypes, inpreventing virologically confirmable dengue disease with a 2-sided 95%confidence interval, wherein the lower bound is more than 60%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects irrespective of serostatus at baseline and 14 to 16years of age, from the first administration of the administrationschedule until 18 months after the second administration of theadministration schedule.
 49. The method of claim 47, providing acombined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 45%, when measuredagainst placebo in a subject population of at least 1,500 or at least2,000 healthy subjects seronegative against all serotypes at baselineand 14 to 16 years of age, from 30 days after the second administrationof the administration schedule until 18 months after the secondadministration of the administration schedule
 50. The method of claim 28which is safe.
 51. The method of claim 50, providing a combined vaccineefficacy against virologically-confirmed dengue with hospitalizationagainst all four serotypes with a 2-sided 95% confidence interval,wherein the lower bound is more than 65%, when measured against placeboin a subject population of at least 5,000 healthy 4 to 16 year oldsubjects irrespective of serostatus at baseline from firstadministration of the administration schedule until 12 to 18 monthsafter the last administration of the administration schedule.
 52. Themethod of claim 28 which is effective.
 53. The method of claim 52,providing a combined vaccine efficacy against all four serotypes, inpreventing virologically confirmable dengue disease with a 2-sided 95%confidence interval, wherein the lower bound is more than 60%, whenmeasured against placebo in a subject population of at least 5,000healthy subjects irrespective of serostatus at baseline and 14 to 16years of age, from the first administration of the administrationschedule until 18 months after the last administration of theadministration schedule.
 54. The method of claim 52, providing acombined vaccine efficacy against all four serotypes, in preventingvirologically confirmable dengue disease with a 2-sided 95% confidenceinterval, wherein the lower bound is more than 45%, when measuredagainst placebo in a subject population of at least 1,500 or at least2,000 healthy subjects seronegative against all serotypes at baselineand 14 to 16 years of age, from 30 days after the last administration ofthe administration schedule until 18 months after the lastadministration of the administration schedule